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 PRESS G Model PRBI-10893; No. of Pages 10 Process Biochemistry xxx (2016) xxx–xxx 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: [email protected] (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, k L a, 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/).

integrated sophorolipid production and gravity separation

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Page 1: integrated sophorolipid production and gravity separation

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ARTICLE IN PRESSG ModelRBI-10893; No. of Pages 10

Process Biochemistry xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Process Biochemistry

journa l homepage: www.e lsev ier .com/ locate /procbio

ntegrated sophorolipid production and gravity separation

en M. Dolman, Candice Kaisermann, Peter J. Martin, James B. Winterburn ∗

chool of Chemical Engineering and Analytical Science, The Mill, The University of Manchester, Manchester, M13 9PL, UK

r t i c l e i n f o

rticle history:eceived 9 November 2016eceived in revised form3 December 2016ccepted 21 December 2016vailable online xxx

hemical compounds:ophorolipid (PubChem CID: 11856871)

a b s t r a c t

A novel method for the integrated gravity separation of sophorolipid from a fermentation broth has beendeveloped, 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 enrichmentof up to 9. Furthermore, the broth viscosity reduction achieved by removal of the sophorolipid phaseenabled a 34% reduction in mixing power to maintain the same dissolved oxygen level by the end of the

eywords:ntegrated separationophorolipidettlingandida bombicolaiosurfactant

fermentation, with a 9% average reduction over the course of the fermentation. Fermentation durationcould 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 highvolume 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

lycolipid

. Introduction

Sophorolipids are microbially produced glycolipid biosurfac-ants, which are rapidly increasing their market share of the 27illion USD global surfactant market [1]. While several yeast strainsre able to synthesize sophorolipids, most research and industrialse is focused on Candida bombicola ATCC 22214, the organism used

n this study [2]. Sophorolipids consist of a hydrophilic sophoroseisaccharide bound to a hydrophobic fatty acid with a typical chain

ength of 16–18 carbon atoms. The fatty acid may be joined byn ester bond to the second glucose monomer, giving a lactonicophorolipid, or joined to only one glucose monomer, giving ancidic sophorolipid due to the unbound fatty acid. These and otherifferences in the fatty acid chain and acetylation of the sophoroseolecules give a range of different structures and properties. Two

ommon structures representing lactonic and acidic sophorolipidsre shown in Fig. 1 [2].

Sophorolipids are produced industrially by a number of compa-ies, who often utilize sophorolipids’ detergent and low foaming

Please cite this article in press as: B.M. Dolman, et al., Integrated so(2016), http://dx.doi.org/10.1016/j.procbio.2016.12.021

roperties in a variety of formulated cleaning products [3]. Theherapeutic properties of sophorolipids have allowed them to beommercialized in anti-dermatitis soap and other body washes,

∗ Corresponding author.E-mail address: [email protected] (J.B. Winterburn).

ttp://dx.doi.org/10.1016/j.procbio.2016.12.021359-5113/© 2017 The Authors. Published by Elsevier Ltd. This is an open access article u

(http://creativecommons.org/licenses/by/4.0/).

and in a cream to reduce oily skin by MG Intobio Co and Soliance.There is ongoing research into potential medical applications ofsophorolipid, with anti-cancer, anti-HIV, antimicrobial and anti-biofilm activity being investigated [4–6]. Sophorolipids also havepotential for use in low cost, high volume applications such asbioremediation and enhanced oil recovery if production costs canbe 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 areroutinely achieved [9–11]. Sophorolipid producing fermentationsbegin with a cell growth phase, which typically lasts until thenitrogen in the media is depleted, at which point the sophorolipidproduction rate increases significantly, if both a hydrophilic and ahydrophobic carbon source are present [12]. The sophorolipid pro-duction phase normally lasts for around 200 h, at which point thedissolved oxygen level in the fermenter cannot be maintained dueto oxygen mass transfer limitation.

This dissolved oxygen reduction is caused by the high viscosityof the sophorolipid produced, meaning the fermentation must bestopped and the sophorolipid recovered [12,13]. It is well knownthat the presence of a separate sophorolipid phase in the bioreactorsignificantly reduces the oxygen mass transfer coefficient, kLa, by

phorolipid production and gravity separation, Process Biochem

both providing a resistance to mass transfer across the air/liquidinterface and increasing the viscosity of the medium, which resultsin oxygen limitation, increased stirring power requirements andnon-homogeneity in the bioreactor [12–14].

nder the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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2 B.M. Dolman et al. / Process Biochemistry xxx (2016) xxx–xxx

F s. A lao ree car

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ig. 1. Molecular structure of common lactonic (left) and acidic (right) sophorolipidf the sophorose in the lactonic sophorolipid, where the acidic sophorolipid has a f

The physical form of sophorolipids is dependent on the con-itions under which they are produced, which directly affecthe proportions of acidic and lactonic sophorolipids produced.ophorolipids typically separate from the fermentation broth as

crystalline material if the lactonic to acidic ratio is high and theydrophobic carbon source concentration is low. The sophorolipidstherwise form a viscous second phase of around 50% sophorolipidnd 50% water, which may sit below residual oil at the surface ofhe broth or sink to the bottom of the bioreactor when agitation istopped [10,15,16].

These properties are commonly exploited at the end of a fermen-ation to give an easy, crude separation of the sophorolipid from theermentation broth, either by crystal decantation, crystal filtrationr decantation of the sophorolipid gel [11,16,17]. These techniquesave not previously been used effectively to recover sophorolipidsuring fermentation.

Industrially, there are a number of costs associated withepeated batch cycles for sophorolipid fermentation, in terms ofowntime between cycles, cleaning costs and the lengthy inocu-

um preparation required for large scale production [18,19]. Therere numerous proposed partial solutions to this problem in the lit-rature. For example, a portion of the broth can be removed andeplaced with fresh media, allowing high productivity to be main-ained for seven 80–130 h cycles, nevertheless removing biomassroportionally to other components [19]. Sophorolipid settling byravity within a fermentation vessel or shake flask has previouslyeen demonstrated for small scale sophorolipid production. Signif-

cant benefits of sophorolipid separation have been shown, with aoubling of the duration of sophorolipid production and little effectn production after 15 min without agitation or aeration demon-trated by Guilmanov et al. [20], and a productivity increase from.38 to 1.89 g l−1 h−1 shown by Marchal et al. [19]. Both studies relyn gravity settling within the fermentation vessel or shake flask,owever, making scale up impractical due to the excessive settlingistances present if this technique were applied at industrial scale.

Effective integrated separation techniques have been developedor other biosurfactant systems, notably foam fractionation forydrophobin proteins, surfactin and rhamnolipids, but there have

Please cite this article in press as: B.M. Dolman, et al., Integrated so(2016), http://dx.doi.org/10.1016/j.procbio.2016.12.021

een no successful scalable attempts at integrated separation forophorolipid production [21–23].

This paper details a novel technique, based on an integratedravity settling column, for removing the sophorolipid phase from

ctonic bond can be seen joining the fatty acid chain to the second glucose monomerboxylic acid to end its fatty acid chain.

the fermentation broth during fermentation, reducing the fermen-tation volume required which allows continued substrate feedingand provides a concentrated product phase. We also demonstrate,for the first time, the application of this technique to extend theproduction phase of a fermentation beyond 1000 h, significantlyincreasing batch production.

2. Methodology

2.1. Fermentation

Sophorolipid was produced by fed batch fermentation using C.bombicola ATCC 22214 in an Electrolab Fermac 320 fermentationsystem (Electrolab, UK) with a 2 l maximum working volume, H:Dof 2, two 55 mm diameter 6-bladed Rushton-type impellers and aninitial 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 platescontained 6 g l−1 yeast extract and 5 g l−1 peptone. The initial con-centration of glucose in all fermentations preculture and agar plateswas 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 agarplates.

Four fermentations were carried out. Fermentation 1 was con-ducted in a conventional manner without separation. Fermentation2 was directly comparable to fermentation 1 except that the in situseparator was used to remove product from the top of the separa-tor. Fermentation 3 was controlled to give a product that separatedfrom the bottom of the separator. Fermentation 4 was carried outfor an extended period of time to demonstrate the potential toutilise the integrated separation to extend the fermentation periodand give higher batch production, and controlled to give separationfrom the surface of the broth. C. bombicola was first transferred fromcryogenic storage (−80 ◦C) onto agar plates, and incubated at 25 ◦Cfor 48 h. Single colonies from these plates were then used to inocu-late 50 ml of medium in 250 ml shake flasks, which were incubatedat 25 ◦C and 200 rpm for 30 h. This inoculum was diluted to an opti-

phorolipid production and gravity separation, Process Biochem

cal density of 20 at 600 nm with fresh media and 100 ml used toinoculate the fermenter.

Fermentations were run at 25 ◦C, and dissolved oxygen was con-trolled to 30% by varying the stirrer speed, whilst maintaining a

Page 3: integrated sophorolipid production and gravity separation

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B.M. Dolman et al. / Process

onstant aeration rate of 1 l min−1. Fermenter pH was controlled to value of 3.5 by the addition of 3 M sodium hydroxide.

The feeding rates of rapeseed oil and glucose were similar forermentation one and two, to facilitate comparison, with differenteeding rates used for fermentation three and four, to give separa-ion of sophorolipid to the top and bottom of the fermenter. Feedingates of oil were modified during the experiments to maintain aow concentration, without limiting production, according to theil concentration in the samples. Glucose concentration was usedo control the relative density of the sophorolipid phase and theulk media to enable effective separation from either the top orottom of the separator, as well as being an important substrateor sophorolipid production. The feed profiles are shown in Fig. 4.

The total sophorolipid produced was calculated by adding theass of sophorolipid in the fermenter and the mass of sophorolipid

emoved from the fermenter using the separator. Contaminationas tested for visually using microscopy and by streak plating.

.2. Separation

Integrated separation of the sophorolipid from the fermentationroth was carried out using an in house built settling column, ashown in Fig. 2. The integrated arrangement of the bioreactor andettling column is shown in Fig. 3 with the settling column sup-orted at an angle of 30◦ from horizontal. The separator was rinsedith 70% ethanol before attaching to the fermenter. Sophorolipid

eparation was carried out intermittently, based on visual observa-ion of a sample taken from the bioreactor. When a significant layerf sophorolipid rich phase could be seen at either the top or bot-om of the sample in the universal bottle within 2 min separationas initiated. Prior experiments indicated that at these conditions

eparation is effective.During separation, which was used intermittently during fer-

entation, sophorolipid rich fermentation broth was continuouslyirculated from the fermenter, through the settling column andack to the fermenter. This was pumped from the fermenterhrough a stainless steel tube with an inlet 20 mm from the bot-om of the fermenter, and then in 8 mm external diameter siliconubing of 1 mm wall thickness to and from the separator. The flowate of media into and out of the settler was controlled to 1 ml s−1

sing Matson Marlow 502 S and 503 U pumps (Watson Marlow,K). This flowrate was based on the results of preliminary experi-ents, giving a residence time in the settling column of 76 s, with

total residence time in the column and tubing of 137 s. In the set-ling column, the sophorolipid phase separates out towards eitherhe top or bottom of the column, depending on the relative densityf the sophorolipid and bulk media. Initially, broth is continuouslyirculated and the sophorolipid product collects in the settling col-mn. When the sophorolipid phase accumulating in the separatoreached 50% of the height of the separator, which typically occurredfter around three minutes of separator operation, the outlet pumpas started to continuously remove the sophorolipid product phase

t a rate controlled between 0.5 and 2 ml min−1, depending on theccumulation or reduction of the sophorolipid phase in the set-ling vessel. The separation was stopped when the separation rateropped below 0.5 ml min−1, until the condition for separation wasgain observed.

.3. Hydrodynamics

To determine the effect of the sophorolipid phase on the agita-

Please cite this article in press as: B.M. Dolman, et al., Integrated so(2016), http://dx.doi.org/10.1016/j.procbio.2016.12.021

ion requirements, the sophorolipid enriched fractions, which hadeen separated from the bioreactor using the separator over theourse of fermentation 2, were pooled and returned to the fer-enter at 308 h after the start of the fermentation over a period of

PRESSemistry xxx (2016) xxx–xxx 3

12 min. The stirrer speed and dissolved oxygen percentages werethen monitored as the fermentation control returned the dissolvedoxygen percentage to the set point. The equation for power numberis shown in Eq. (1);

P = Np�n3D5 (1)

where P is power, Np is power number, � is density, n is stirrer speedand D is stirrer diameter.

Due to identical fermenters being used and assuming the den-sity is constant (as density changes during the fermentation arerelatively small), the power input as a function of power numbercan be calculated during the fermentation. The power input wasintegrated over time to determine the power input for the wholefermentation, with the power input for fermentations equated untilthe time point of the first separation.

Separation performance was measured in terms of enrichmentand recovery, which are defined in Eqs. (2) and (3);

enrichment = CpCf

(2)

recovery (%) = Cp × VpCf × Vf

× 100 (3)

where Cp is the sophorolipid concentration in the product, Cf is theproduct concentration in the fermenter before separation, Vp is thevolume of product phase recovered, and Vf is the initial volume ofliquid in the fermenter.

2.4. Analytical techniques

For all analysis, 5 ml of broth was removed from the bioreactor or5 ml product was taken from the sophorolipid collection vessel con-nected to the separator. The sample was centrifuged at 5000 rpmfor 5 min using a Sigma 6–16S centrifuge (Sigma laboratory cen-trifuges, Germany) and the glucose in the supernatant quantifiedusing 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 sophorolipidwere then applied to the whole sample, with oil concentra-tion measured gravimetrically from the hexane extraction, andsophorolipid measured gravimetrically from the pooled ethylacetate extracts[24–26]. These extracts were dried to constantweight in weighing dishes at ambient temperature for 30 h.

Cell growth was determined by both dry cell weight and opticaldensity measurement. After the aforementioned hexane and ethylacetate extractions, 8 ml distilled water was added to the remainderof the sample in the centrifuge tubes, which were then centrifugedat 8000 rpm for 10 min. The supernatant was discarded and theresulting cell pellet was resuspended in 8 ml distilled water. Thiscell suspension was transferred to drying trays, which were driedto constant weight at 90 ◦C in a drying oven. Optical density wasused as a proxy for dry cell weight when diluting the inoculum, ata wavelength of 600 nm.

The structure of the sophorolipids produced was determinedwith negative electrospray ionisation mass spectrometry, usingan 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,

phorolipid production and gravity separation, Process Biochem

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 (TAInstruments, USA).

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F w, side view and end view are shown, all dimensions are internal dimensions shown inm

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Table 1Key metrics for all sophorolipid producing fermentations. Fermentation 2, 3 and4 used sophorolipid separation, with fermentation 1 included for comparison. Allmetrics are the result of unique fermentations. Due to volume changes caused bysubstrate 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 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) 325 325 270 623

ig. 2. Diagram of custom built sophorolipid separator used for this study. Plan viem..

. Results and discussion

A novel gravity sophorolipid separation technique was success-ully applied to three fermentations. Results from one fermentation

ithout separation, fermentation 1, are presented alongside resultsrom three fermentations with separation, fermentations 2, 3 and. Fermentations 1 and 2, without and with integrated separa-ion respectively, are directly comparable, to allow evaluation ofhe effect of separation, but due to feeding rate control to enableophorolipid to be recovered from the bottom of the separatorermentation 3 is not directly comparable. Fermentation 4 wasntended to demonstrate an extended fermentation, and so is alsoot directly comparable. The separation was run periodically inll fermentations, when sufficient sophorolipid phase had accu-ulated, with the majority of the available sophorolipid phase

eparated.

.1. Fermentations

Fig. 4 shows the feeding rate of substrates for the fermentationsresented, which enabled control of the sophorolipid phase to sep-rate from the surface or bottom of the separator, whilst also beingn important parameter for sophorolipid production. The progressf the fermentations over time is presented in Fig. 5, and the keyetrics from these fermentations are presented in Table 1.

Fig. 5 shows the progress of fermentation 1, without separation,nd fermentations 2, 3 and 4, during which sophorolipid productas separated from the fermentation broth. In fermentation 2 and

, sophorolipid was recovered at the top of the integrated gravityeparator, and in fermentation 3 the sophorolipid was collectedrom the bottom of the separator. In fermentation 2, separation

as carried out at 111, 184 and 261 h, in fermentation 3 at 72, 281,55 and 376 h and in fermentation 4 at 86, 111, 160, 186, 232 and40 h. No separation of the sophorolipid phase was carried out inermentation 1.

Please cite this article in press as: B.M. Dolman, et al., Integrated so(2016), http://dx.doi.org/10.1016/j.procbio.2016.12.021

In fermentation 2, the glucose concentration initially rose, andemained above 50 g l−1 for the majority of the fermentation, whiched to the sophorolipid rising to the surface of the fermentationroth without agitation. A high glucose concentration throughout

Total dry cell weight (g) 16.1 21.0 16.5 32.1Yield product on biomass (g g−1) 20.2 15.5 16.4 19.4

fermentation 4 meant the sophorolipid was also separated from thetop 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 couldbe observed to settle in a sample bottle within 2 min. Settling wasnot possible after this until 283 h due to the high residual glucoseconcentrations caused by pulse glucose feeding, which was used toensure good sophorolipid production. Whilst the relative density ofthe sophorolipid phase and the broth also depend on other factors,a glucose concentration of 50 g l−1 tends to represent a thresholdof sophorolipid phase separation to the surface or the bottom ofthe separator. This is because higher glucose concentrations leadto higher media densities, meaning the sophorolipid phase is rela-tively less dense the higher the glucose concentration. After 283 hthe glucose concentration had dropped sufficiently for settling tobe used again. Lower glucose feeding could enable sophorolipidsettling throughout the fermentation, though this might impactsophorolipid production.

Fermentations 1 and 2, which were identical apart from the

phorolipid production and gravity separation, Process Biochem

application of integrated sophorolipid separation in fermentation2, each produced 325 g sophorolipid, with 270 g sophorolipid pro-duced during fermentation 3. The dry cell weight production, of16–21 g, and the yields of product on substrate, of 0.33-0.39 are

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F eparaf duct is e den

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ig. 3. Integrated fermentation system. Bioreactor is shown on the left, with the srom the bioreactor into the separator, and recirculated back to the bioreactor. Proophorolipid phase density higher than fermentation broth. (b)− sophorolipid phas

n line with other results in the literature, as are the values forotal sophorolipid produced [9,25]. The use of the separator appearso have little effect on the total production of sophorolipids overhe same time period, with identical values recorded for the twoomparable fermentations.

In fermentation 2 with separation, 21 g of biomass were pro-uced, more than the 16 g of cell biomass produced in fermentation, with product yields on biomass of 15.5 g g−1 and 20.2 g g−1

espectively. The reason for the reduction in product yield oniomass in fermentations 2 and 3 is not known, but with furtherptimisation fermentation 4, with separation, reached a productield on biomass of 19.4 g g−1.

The highest working volume reached during a fermentation dic-ates the overall fermenter volume required, and the high feedingates used during sophorolipid producing fermentations lead to aarge increase in the working volume required over time, much of

Please cite this article in press as: B.M. Dolman, et al., Integrated so(2016), http://dx.doi.org/10.1016/j.procbio.2016.12.021

hich is only required in the later stages of the fermentation.The use of integrated sophorolipid separation in fermentation

decreased the fermenter working volume required by removing23 ml broth from the fermenter using the separator. This meant

tor in the center, and the product collection vessel on the right. Broth is pumpeds pumped from the separator into the collection vessel. This can be used for; (a) −sity lower than fermentation broth.

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 capitalcosts. The corresponding maximum volume for fermentation 3 was1350 ml, but was not directly comparable due to differences infeeding rates between fermentations 2 and 3.

The overall productivity of fermentations 1 and 2 were 1.07 gl−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 ofsophorolipid production slowed dramatically when the oil wasdepleted after around 80 h in fermentations 1–3, reducing from 2 gl−1 h−1 to 0.6 g l−1 h−1 and increasing the oil feed rate did not returnthe productivity to the previous level. Many studies have demon-strated a fairly constant production rate throughout a fermentationuntil the point at which the fermentation had to be stopped due todissolved oxygen limitation, and with improved feeding control in

−1 −1

phorolipid production and gravity separation, Process Biochem

fermentation 4, a productivity of 2.0 g l h was maintained until158 h [17] [12].

In fermentation 4, fermentation was continued past the pointat which fermentations usually have to be stopped due to product

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F y. Gluf tationv

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3

s

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ig. 4. Feeding profiles of glucose and rapeseed oil for all fermentations in this studermentation 2 (dotted, a), fermentation 3 (b) and fermentation 4 (c). (For interpreersion of this article.)

ccumulation, and run for a total of 1023 h. This enabled the produc-ion of 623 g sophorolipid from a 1 l initial broth, which comparesavourably to the highest previous reported titers of around 400 g−1, and clearly demonstrates the capacity of integrated separationo extend the time period of sophorolipid production in fermenta-ion. This could lead to a dramatic improvement in overall processroductivity, by reducing the proportion of time spent in inoculumreparation, biomass production and cleaning.

.2. Separation

The separation results achieved in fermentations 2, 3 and 4 arehown in Table 2.

During fermentations 2, 3 and 4 with integrated separation, theajority of the sophorolipid was removed from the fermentation

roth, with 86% of the total sophorolipids produced separated inermentation 2, 74% separated in fermentation 3 and 65% separatedn fermentation 4.

Almost no cells and only 8 g of oil were removed by the separa-ion over the course of fermentation 3, determined by gravimetricnalysis as for fermentation samples. This is because the rate of

Please cite this article in press as: B.M. Dolman, et al., Integrated so(2016), http://dx.doi.org/10.1016/j.procbio.2016.12.021

ettling of the cells was much slower than the settling of theophorolipid product, and oil rose to the surface of the separa-or rather than sinking to the bottom of the separator with theophorolipid. Cell removal was also negligible in fermentation 2,

cose (blue), rapeseed oil (green) and total (red) shown for fermentation 1 (solid, a) of the references to colour in this figure legend, the reader is referred to the web

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 cellswere removed during fermentation 4, which represents a smallproportion of the total biomass, 32.1 g.

The enrichment varied significantly between separations atdifferent time points, from 2.5 to 9. This is largely due to thesophorolipid concentration present in the fermenter before theseparation, as there was little variation of the concentration in thesophorolipid enriched product fraction, of approximately 550 g l−1.In fermentation 2, when the sophorolipid phase was separated fromthe top of the settler, a total of 68 g oil was recovered along withthe sophorolipid during the fermentation, which was the primaryreason for the variations in the concentration of the sophorolipidphase. There is little scope to improve the product phase concentra-tion above that demonstrated in fermentation 3 using the currenttechnique, however, with an increased fermenter volume, the sys-tem could operate at lower initial sophorolipid concentrations andso give an improved enrichment.

Sophorolipid recoveries would be expected to improve sig-nificantly as fermentation scale is increased; in laboratory scale

phorolipid production and gravity separation, Process Biochem

experiments the separation had to be stopped as the layer ofsophorolipids at the bottom/top of the settling column became toolow, to prevent the media and cell phase being entrained in theproduct 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 thisarticle.)

Table 2Separation results for fermentation 2, fermentation 3 and fermentation 4. All metrics are the result of unique fermentations, with the application of the novel integratedgravity separation developed in this study.

Time (h) Sophorolipidrecovered(g)

Sophorolipidconcentration(g l−1)

Total sophorolipidpresent(g)

Sophorolipid productconcentration(g l−1)

Enrichment Recovery at timepoint(%)

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 87Total 280.1 86Total oil removed by separation (g) 8 Total cells removed by separation (g) below detection limit

Fermentation 371.5 16.8 103.7 128.4 582.9 5.62 13281 79.5 168.3 238.2 654.1 3.89 33355 59.2 109.2 175.3 616.9 5.66 34376 45.5 106.3 148.9 638.7 6.01 31Total 201.0 74Total oil removed by separation (g) 68 Total cells removed by separation (g) below detection limit

Fermentation 485.6 93.1 152.7 229.1 443.5 2.90 40111.3 53.5 97.1 127.8 504.8 5.20 42159.7 61.9 129.6 186.8 538.1 4.15 33185.8 57.9 72.5 105.3 567.8 7.83 55231.5 48.9 51.7 68.3 465.7 9.01 72

ms

540.3 89.0 124.7 191.3

total 404.3

Total oil removed by separation (g) 20

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ust be recycled back to the fermenter, would be identical irre-pective of fermenter volume while maintaining a given size of

312.2 2.5 4765

Total cells removed by separation (g) 2.6

phorolipid production and gravity separation, Process Biochem

separator, hence a larger total fermenter volume would lead to alarge reduction in residual sophorolipid concentration.

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8 B.M. Dolman et al. / Process Biochemistry xxx (2016) xxx–xxx

F ygen

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ig. 6. The effect of sophorolipid separation on agitation requirements. Dissolved oxhe dissolved oxygen concentration after sophorolipid rich fractions separated duri

Whilst the separator was designed for continuous operationydrodynamic considerations, in particular the turbulence causedy inlet and outlet disturbances which become more significantith decreasing scale, mean it could not be scaled down further toatch the sophorolipid production rates achieved in the 1 l initialorking volume fermenter. At the scale presented in this paper,

he separation occurred at a rate of around 2 ml min−1, or around g min−1 sophorolipid, 30–150 times greater than the productionate. This separation rate makes it suitable for use with a separa-or of 30–60 l volume for continuous separation of the sophorolipidroduced. Product recovery rate is expected to scale proportionallyo the volume of the separating column, so for a new settler designolume can be increased while maintaining or reducing the ratio ofnertial to viscous forces, i.e. the Reynolds number. Residence timehould be increased proportionally to diameter increase, to allowhe same quantity of sophorolipid to settle or float to the surface athe same settling/rising rate. The system could easily be connectedor steam in place sterilization at industrial scale.

This is the first study to present the design and demonstratehe feasibility of a separation system for sophorolipid productionhat could be continuously applied, having been shown in this

anuscript to separate sophorolipid whilst production continues

Please cite this article in press as: B.M. Dolman, et al., Integrated so(2016), http://dx.doi.org/10.1016/j.procbio.2016.12.021

n the bioreactor for periods of more than one hour. It is also therst integrated separation system applicable to large scale fermen-

ation, because it does not rely on separation within the bioreactor,nd therefore the first to enable an extended sophorolipid produc-

ig. 7. Mass spectrum of sophorolipid from the end of fermentation 2. Key peaks of C18:87.

and stirrer speed profiles showing the increase in stirrer speed required to maintainmentation 2 returned to fermenter in fermentation 2 at 308 h.

tion period at scale. The reduced bioreactor volume, and reducedstart up and cleaning costs of this system could significantlyimprove the economics of sophorolipid production by increasingthe total sophorolipid produced per batch. This would make bulkapplication, such as for enhanced oil recovery, a more realisticproposition.

Other glycolipid biosurfactants, notably rhamnolipids and man-nosylerythritol lipids (MELs), as well as many other bioproductsincluding those used as biofuels, may also form a separate, insol-uble, phase in a fermentation broth, and so the gravity separationtechnique presented in this paper could likely also be applied tothese systems [27,28].

3.3. Effect on hydrodynamics and mass transfer

Fig. 6 shows the dissolved oxygen level and stirrer speed at theend of fermentation 2, capturing the addition of the sophorolipidrich fractions which were removed by separation during fermen-tation 2 and subsequently pooled, and added to the bioreactor at308 h. The presence of this sophorolipid phase effectively reducedthe volumetric oxygen transfer coefficient, kLa, in the fermenter,due to its high viscosity of around 0.5 Pa.s, resulting in an increase

phorolipid production and gravity separation, Process Biochem

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 rpmwas required to maintain the desired dissolved oxygen level whenthe sophorolipid product separated over the whole fermentation

1 diacylated acidic sophorolipid at m/z 705 and C18:1 lactonic sophorolipid at m/z

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as added. Given the high viscosity of the sophorolipid phase, its possible that turbulence was not attained even at the highergitation rate; in this instance a still higher agitation rate woulde required for proper dissolved oxygen control throughout theioreactor, leading to larger savings than calculated.

The mixing Power number in the turbulent regime is typicallyonstant, so changes in impeller speed have a cubic impact on theixing power. Removing the sophorolipid phase resulted in a 13%

ecrease in impeller speed and a 34% decrease in mixing powerequirement by the end of the fermentation.

The relative power requirements over fermentation 1 and 2ere also compared, with an 18% reduction in power input from the

ime point of the first separation observed, and a 9% improvementhen the whole fermentation is taken into account.

There would be some increased power consumption from theumping of the fermentation broth between the separator and the

ermenter, but it is expected this would be significantly smallerhan the agitation power at scale, as relatively low pumping flowates are used. If these power reductions can be achieved at indus-rial scale, they can give significant cost savings.

.4. Sophorolipid structure

Mass spectra of sophorolipid samples were taken to determinehe sophorolipid structures produced during the fermentation,ith the mass spectrum of the sophorolipids taken from the end

f fermentation 2 shown in Fig. 7. The main peak at m/z=705epresents a diacylated acidic C18:1 sophorolipid, with the peakt 687 representing a diacylated C18:1 lactonic sophorolipid [29].here were some differences in the ratios of peak heights betweenophorolipid taken from the settler and sophorolipid taken fromhe fermentation broth immediately before, suggesting this tech-ology may have potential to act as a crude separator of differentophorolipid forms.

Recent research has revealed the enzyme responsible for lac-onisation of acidic sophorolipids, enabling the use of geneticngineering of the genome of Starmerella bombicola, and robustroduction and purification techniques to yield a 98% pure lactonicr acidic sophorolipid, making the application of sophorolipid inedicinal or personal care products much more likely [3]. Comple-entary to the advances in genetic engineering and purification,

his work makes significant progress in solving some of the complexngineering challenges involved with sophorolipid production, giv-ng potentially dramatic reductions in production cost and makingarge scale application of sophorolipid more feasible.

. Conclusions

A novel method for the integrated separation of sophorolipidrom a fermentation process has been developed. The design of theystem overcomes the production and processing difficulties asso-iated with in situ (i.e. in the fermenter vessel) gravity separationf sophorolipids for scale up, and with a separator residence timef less than two minutes the process seemed to have no impact onurther sophorolipid production by the cells. A sophorolipid phasean be removed from the fermentation broth if the product phasead higher or lower density than the media, with enrichments ofp to 9, an overall recovery of 86%, and up to 404 g of sophorolipidecovered from the fermentation broth.

We demonstrated an 11% decrease in bioreactor working vol-

Please cite this article in press as: B.M. Dolman, et al., Integrated so(2016), http://dx.doi.org/10.1016/j.procbio.2016.12.021

me requirement when using the separator, due to the removal ofhe sophorolipid product phase. Optimised feeding rates and settlersage could further reduce the volume requirement. By using theeparator, the fermentation could be run for 1023 h, and produce

[

PRESSemistry xxx (2016) xxx–xxx 9

623 g sophorolipid from a 1 l initial batch, reducing the number offermentations required for a given product mass.

An 18% average reduction in stirrer power was demonstratedover the course of a fermentation once sophorolipid separation wasinitiated, which translates to around 9% over the entire fermenta-tion, with a 34% decrease in power input shown by the end of thefermentation.

The integrated separation system presented in this paper hasbeen developed for sophorolipid separation, but could equally beapplied to the production of other insoluble bioproducts, in par-ticular mannosylerythritol lipids. With correct scaling up, it isanticipated that the advantages this system offers will lead to adramatic improvement of the economics of sophorolipid produc-tion.

Acknowledgements

Sara Bages Estopa is acknowledged for her invaluable commentson 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 apatent 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) andthe EPSRC DTA fund, which enabled this work to be conducted.

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