Abstract

Environmentally friendly, microbially produced surfactants known as biosurfactants have

recently seen an explosion in commercial activity and interest due to a reduction in the cost of

production, though these costs still limit biosurfactant use in bulk applications. These high

production costs are primarily the result of the typically low productivities of large scale

biosurfactant production processes and hence the large production volumes required, as well

as process engineering challenges related to the nature of the biosurfactant produced. This

review details the use of integrated separation technologies, primarily gravity, membrane and

foam fractionation separations, in integrated biosurfactant producing fermentations, to tackle

these difficulties. An analysis of the scalability of the available technologies and the expected

impact on process economics is presented, demonstrating the potential utility of integrated

separation processes for bringing biosurfactants into mainstream commercialisation.

Keywords

Integrated separation, fermentation, biosurfactant, gravity separation, foam fractionation,

membrane separation

Chemical compounds

Sophorolipid (PubChem CID: 11856871)

Surfactin (PubChem CID:443592)

Rhamnolipid (PubChem CID: 5458394)

Mannosylerithritol lipid

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1. Introduction

Surfactants are a class of molecules critical to the functionality of both cleaning and

formulated products with a $32-36 billion annual market [1, 2]. The vast majority of the

surfactants currently used in these market sectors are either oil derived or derived from

natural products via chemical reactions. Many surfactants used in agriculture are toxic to

humans and aquatic life and most of those used in personal care are irritants [3]. Many major

chemical companies are looking for alternatives that do not have these toxic or irritant

properties. This drive is illustrated by an open innovation competition run by Nouryon and

Unilever, which describes that for some of their applications “reducing toxicity remains an

urgent challenge” for which innovative solutions are needed [4].

Microbial biosurfactants are surfactants produced by microorganisms, typically from

carbohydrates and/or vegetable oils. Biosurfactants often have an enhanced environmental

profile and reduced toxicity, as well as superior performance in many applications compared

to oil based surfactants, with biosurfactant properties being reviewed extensively elsewhere

[5-10]. Due to their desirable properties, interest in these biological molecules is therefore

increasing substantially, with multibillion-dollar chemical companies such as Evonik

beginning production, and a rapid rise in the rate of patenting related to these molecules, as

illustrated in Figure 1.

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Figure 1 - Annual patents related to sophorolipid production and application, 2000 to 2016.

The price of chemical surfactants such as sodium lauryl sulphate in bulk applications tends to

fall in the range $1-2 kg−1, and for specialty surfactants, such as amino acid based surfactants,

in the range of $3-4 kg-1. The sales price of sophorolipid, the cheapest and most widely

available microbial biosurfactant, has recently been published at $ 34/kg active matter [11].

This price is several times higher than the price of typical specialty surfactants, highlighting

the significant need to reduce the production cost for biosurfactants in order to make their use

in a broader range of bulk applications viable and to overcome the industry challenges

associated with standard chemical surfactants.

Biosurfactants are produced by fermentation and extensive industrial and academic research

has gone into increasing the productivity, titer and yield of the fermentation process, as well

as into the use low cost raw materials and the utilization of waste for production, that are

reviewed extensively elsewhere [5, 12-15]. Several challenges and opportunities are present

in biosurfactant production due to the surface active nature of the product, particularly foam

formation and the potential for the production of a separate product phase. These challenges

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give rise to the opportunity to develop and apply several techniques with potential for

recovering the biosurfactant product. This review provides an overview of the current

challenges faced in extracellular biosurfactant manufacture and discusses how innovative

integrated separation techniques are able to address these issues, with an analysis of how such

techniques could assist in bringing biosurfactants to market and also expand biosurfactant

application to new market sectors. More specifically, an overview of the specific challenges

associated with biosurfactant production is given in Section 2, followed by an analysis of the

efforts made to overcome and exploit each of these challenges to give an integrated

production and separation process in Section 3. In Section 4 the scale up challenges and

potential scalability of each of these techniques are discussed and, in Section 5, the potential

economic impact of the application of each of these separation techniques is discussed. The

focus of this review is extracellular biosurfactants, as to the best of the authors knowledge

these are the only biosurfactants for which integrated production and separation has been

applied.

2. Challenges in extracellular biosurfactant production

A brief overview of a generic extracellular biosurfactant production process is shown in

Figure 2. Typically, a cell growth period with excess nitrogen source as well as carbon source

is usually followed by nitrogen exhaustion, which is often the trigger for biosurfactant

production [16, 17]. At this point continuous carbon substrate addition can be used in fed

batch fermentation to maximise production rates and titer [18, 19]. Biosurfactant production

processes are typically aerobic, with a large biological oxygen demand leading to the need for

high oxygen mass transfer rates, consequently high agitation and aeration rates are required.

After the fed batch, production reaches some sort of limitation and end point, due to product

inhibition, reaching the maximum vessel working volume, mass transfer limitation etc. The

fermentation is then stopped, the biosurfactant product recovered from the fermentation broth

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and the whole process repeated for further production. During the production period foaming,

bioreactor volume limitation and biosurfactant product viscosity can all have a significant

detrimental effect on bioprocess performance, all of which are discussed in more detail in

section 2.1-2.3.

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2.1. Foaming

For many surfactant applications foamability and high foam stability are extremely desirable

properties. Consequently, when biosurfactants with a high propensity for foaming and able to

generate stable foams are produced in an aerated and agitated bioreactor, foaming, often

uncontrolled, is a significant processing issue [20]. Traditional methods to combat foam

formation, such as antifoam addition and mechanical foam breakers, often prove insufficient

to prevent foaming problems and can lead to batch failure, loss of containment and associated

loss of production [19].

Various techniques have been proposed to overcome foaming issues, including running

fermentations without sparging air, which has been shown to prevent foaming in surfactin

fermentations, but also reduces surfactin production as an unwanted side effect [21]. It is

likely that with an oxygen overlay this limitation could be reduced, but oxygen transfer

would remain a key limitation for these fermentations. Achieving oxygen transfer through the

use of membranes can also eliminate foaming and has been shown to be effective at

providing sufficient oxygen for production at laboratory scale, but would be difficult and

expensive to apply at an industrial scale due to the high membrane surface area requirement

and the fouling associated with operating a membrane in contact with cells [22]. Uncontrolled

foam generation can lead to product overflow, which increases the chance of contamination

and is unfeasible in a production scenario, but the application of controlled foaming leads to

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Biosurfactant

recovered and process

repeated

Feeding of substrate

continued until

limitation reached -

product, oxygen mass

transfer etc.

Nitrogen limitation

and excess substrate

trigger biosurfactant

production

Hydrophilic and/or

hydrophobic carbon

source and nitrogen

source provided for

cell growth

Figure 1 - General process overview for microbial biosurfactants.

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the generation of a surfactant enriched foam which can subsequently be separated using foam

fractionation.

2.2. Product viscosity

Many biosurfactants have been reported to crystallise or form a second, viscous, liquid phase

during fermentation. It has been suggested that the formation of this second phase is the

reason for the apparent lack of product inhibition in these fermentations, as the cells receive

much less exposure to products which exist in a second phase than to products in the

aqueous, cell containing fermentation broth, phase [23]. The formation of this second phase,

whilst preventing direct product inhibition, results in increased resistance to mass transfer and

increases the agitation/aeration power requirements substantially, primarily due to the

viscosity of the product. In sophorolipid production, this increase in agitation power

requirement was estimated to be 30% by the end of the fermentation [24]. Heterogeneity in

the bioreactor also leads to substantial sampling difficulties, with measured product

concentration variation approaching 100% between the final measurement in a fermentation

and that of the whole broth after the fermentation [23]. On occasion, fermentations reach an

endpoint and have to be stopped due to the high viscosity of the product phase with as little as

54 g l−1 of sophorolipid produced, compared to the 100s of g l−1 which are otherwise

achievable [25]. An image of a bioreactor in operation for the production of sophorolipids

without product separation taken by the authors is shown in Figure 3, illustrating the

heterogeneity in the bioreactor. Similar heterogeneity has been observed during

mannosylerythritol lipid (MEL) producing fermentations, with 27% of the MEL produced

attaching to the vessel walls [19], cells and biosurfactant have also been shown to adhere to

the vessel walls in trehalolipid fermentations [26], giving rise to further product recovery

challenges. Attempts to reduce the effects of product accumulation and viscosity have

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included preventing product expression during the cell growth phase [27], as well as

integrated product recovery which is discussed in Section 3.

Figure 3 - Sophorolipid product accumulation and bioreactor heterogeneity during fermentation.

2.3. Bioreactor filling

An often-overlooked aspect of fed batch processes is the additional bioreactor headspace or

dead volume required at the beginning of a fermentation run to allow for subsequent feeding

during the fed batch stage. The high feeding rate required for biosurfactant producing

fermentations results in the need for a relatively large initial dead volume, and often the

fermentation has to be ended when the bioreactor volume capacity is reached. This is

particularly pertinent to sophorolipid producing fermentations where some of the highest

feeding rates of >4 ml l−1 h−1 are necessary [28]. Despite not being widely discussed in the

literature this is, in the authors opinion, the most significant bottleneck which must be

addressed in order to increase sophorolipid productivity and titer, especially in the case where

large-scale bioreactor facilities and production capacity are already in place. Some attempts

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Cells and media

Viscous sophorolipid rich phase

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have been made to mitigate this capacity issue though often the authors of these works don’t

specifically identify that such a solution is being provided. These partial solutions include the

feeding of solid glucose powder rather than a glucose solution to the bioreactor, which would

be challenging at industrial scale and lead to an increased likelihood of contamination [29].

3. Integrating biosurfactant production and separation

Integrated production and separation has primarily been investigated as a method to remove

volatile (e.g. terpenes), easily degraded (proteins) or toxic (e.g. ethanol) products from the

fermentation broth [30, 31]. Integrated production and separation is now also being

investigated for several processes where mass transfer limitations and the need to uncouple

production and product removal are important considerations [31].

A variety of techniques have been proposed for integrated separation processes, including

adsorption, solvent extraction, gas stripping and foam fractionation [30, 32-34]. Whilst more

than 250 integrated product recovery processes have been described in the literature to date,

these have primarily been developed at lab scale and very few have been successfully

commercialised, highlighting the difficulties involved in scaling integrated product recovery

systems to industrially relevant volumes [30, 35].

Some of the main reasons for the difficulty involved in scale up of processes with integrated

product recovery are the harsh separation conditions that are often required, along with the

incompatibility of much of the separating equipment used with cell biomass, due to issues

related to fouling, biofilm formation and contamination. Biofilm formation is particularly

relevant for systems where packing, such as the use of beads in an adsorption column, or

membranes are involved because of difficulties in cleaning. These problems necessitate a cell

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separation system before product separation can occur, making such processes prohibitively

complex and expensive.

The commonly used downstream processing techniques used to separate biosurfactants from

the fermentation broth and the potential of these techniques for application in integrated

separation are shown in Table 1. In many cases, these techniques have been applied primarily

for biosurfactant quantification and sample generation and an understanding of how these

techniques can be scaled up or applied industrially is lacking [36].

As can be seen from Table 1, the three technologies which have been used for biosurfactant

separation with good potential for integrated separation are foam fractionation, membrane

separation and gravity separation. All of these techniques have been applied for integrated

separation at lab scale as a minimum and are discussed in more detail in Sections 3.1, 3.2 and

3.3.

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Table 1- Techniques for separating biosurfactants and their potential for use in integrated production and separation systems, adapted from [37].

Separation technique Biosurfactant Potential for use in integrated separation

Solvent extraction Sophorolipids, MELs, rhamnolipids, trehalolipids

Minimal (with solvents used), cell death

Adsorption Any biosurfactant Potential to use with a cell separation step before adsorption, cells would foul column.

Membranes/filtration Lipopeptides, glycolipids Successfully demonstrated, but potential for use with dissolved biosurfactants. Precipitated biosurfactants may cause fouling.

Foam fractionation Rhamnolipids, lipopeptides, sophorolipids, MELs

Successfully demonstrated

Gravity separation (no salt)

Sophorolipids, MELs Successfully demonstrated.

Gravity separation (salting out)

Sophorolipids, MELs, rhamnolipids Difficult to apply without cell damage

Gravity separation (acid precipitation)

Surfactin, rhamnolipid Difficult to apply without cell damage.

Gravity separation (crystallisation)

Sophorolipids, cellobioselipids Proof of principle verified.

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3.1. Foam fractionation

Foam fractionation utilises the surface activity of biosurfactants for separation, exploiting the

propensity of biosurfactants to accumulate at the air-water interface. Many biosurfactants of

commercial interest will accumulate rapidly at a freshly generated air-water interface, i.e. an

air bubble surface, resulting in the formation of a stable foam in highly agitated, aerated

bioreactors. If this foaming is uncontrolled foam will fill the bioreactor headspace and, in the

simplest foam fractionation systems, a biosurfactant enriched foam overflows from the

bioreactor via the air outlet in an uncontrolled manner and is collected in an overflow bottle

[38]. This basic and non-scalable system has been improved through the addition of a

fractionation column in place of a condenser on the bioreactor to give improved enrichment

[39]. To decouple the fermentation and foam fractionation column operating conditions,

further improve the process and hence be able to optimise the production and separation

independently, a recirculation loop sending biosurfactant rich fermentation broth from

bioreactor to foam fractionation column and returning the biosurfactant depleted broth to the

bioreactor has been used by several authors, giving a controlled foam separation [32].

Foam fractionation has been applied or observed as an integrated separation technology for

surfactin, rhamnolipids and MELs, and sophorolipid enriched overflow has been observed for

sophorolipid fermentations by the authors under certain conditions [19, 40]. There has been

sufficient industrial interest in foam fractionation technology for several patents for both

recirculating foam fractionation and a novel bioreactor for foam fractionation to be filed [41,

42].

Foam fractionation can give very high product enrichments, reported at over 50 for surfactin,

hydrophobin proteins and rhamnolipids [32, 40] and tends to be most effective for systems

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with a dilute product, concentrating products from initial broth concentrations in the order of

10s mg l−1 to final foamate concentrations of 100s mg l−1 or even reaching g l−1 [32]. At a

surfactant concentration characteristic of the system and dependent on the adsorption

kinetics, the bubble surface becomes saturated and the surfactant concentration in the foam

cannot increase irrespective of the surfactant concentration in the broth, though in dilute

systems the mass transfer rate onto the bubble system may be the factor limiting surfactant

concentration in the foam.

A second, more important, limitation, is the usual requirement for high agitation and aeration

in the fermentation broth, which results in uncontrolled foam leaving the bioreactor which

maintains the broth biosurfactant concentration below the threshold value required for

foaming [26]. Even when using integrated foam recovery in a foam fractionation column

coupled to a bioreactor, the threshold surfactant concentration for overfoam is often reached

in the bioreactor, when the bioreactor is fully aerated. This would effectively mean a more

intensive aeration regime in the foam fractionation column would be required to prevent

overflow, which decreases the enrichment of the product from the foam fractionation column

[21]. To overcome this challenge, a system design where the bioreactor is unoxygenated, and

the cells receive oxygen only in the foam fractionation column, has been proposed and shown

to prevent oxygen limitation [21]. This relies on a broth residence fraction in the separator of

a similar order to the bioreactor and a very low cell density, and hence would be unsuitable

for intensified high cell density processes. Oxygen transfer across a membrane can also be

used to oxygenate the broth, albeit at a large expense and with significant potential for

fouling [21, 22].

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Another important aspect of integrated separation processes is the effect on cells. It has been

demonstrated that for foam fractionation of surfactin produced by B.subtilis, cells are

concentrated in the broth after foaming, remaining in the bioreactor rather than leaving in the

foam [43]. In addition, the cells subjected to foaming, when subsequently used as an

inoculum, outperformed unfoamed cells for both growth and surfactin production, albeit

marginally [43]. Other studies for hydrophobin proteins have suggested reduced bioreactor

cell concentrations when using foam fractionation result in a lower productivity compared to

fed batch processes using antifoam without integrated foam fractionation [38].

High enrichments have been demonstrated for biosurfactants using integrated foam

fractionation in a number of studies, but the concentration in the foamate is still relatively low

at below 20 g l−1 for all the systems of which the authors are aware, making efficient

separation difficult. When these challenges are combined with the relatively low

productivities and titers demonstrated for these systems of <0.1 g l h-−1 and <10 g l−1,

integrated foam fractionation remains some way from providing an efficient integrated

production and separation method for biosurfactants. Productivity and titer limitations for

these fermentations, and challenges associated with the threshold concentration for

overfoaming need be overcome in order to develop a foam fractionation system that can be

applied industrially at a reasonable cost.

3.2. Membrane separation

Membrane separation relies on selective permeability of a membrane to components of

interest in a fermentation broth and a pressure gradient to drive flow across the membrane. A

microfiltration membrane can be used to initially recover cells which are returned to the

bioreactor in the retentate, whilst the biosurfactant rich and cell free permeate is passed to an

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ultrafiltration membrane [22, 44]. The ultrafiltration membrane is permeable to water and

other small molecules in the fermentation broth but keeps the biosurfactant as a retentate.

Membrane separation has been applied for surfactin, wild type sophorolipids, acidic

sophorolipids and ns bola sophorolipids [15, 22, 44]. These studies have predominantly been

performed at laboratory scale, but there is clear industrial interest with Ecover having

attempted to scale up the technology to pilot plant scale [44]. Whilst membrane separation

with wild type, insoluble, sophorolipids has been attempted, the focus of membrane

separation has been on non-phase separating, soluble biosurfactants, which are harder to

recover with other methods and cause less membrane fouling than phase separating

biosurfactants, making membrane separation straightforward.

Membrane separation has the advantage of enabling the fermentation and separation to be

entirely decoupled, after an initial cell separation with a microfiltration membrane, which can

easily be applied with soluble biosurfactants [15]. Whilst the use of an additional cell

separation step adds some cost to the process, the cells can easily be recycled back to the

bioreactor and are not subjected to further damaging conditions that are used in the

subsequent separation step.

Membrane separation has been shown to give performance improvements for biosurfactant

production. An ultrafiltration based integrated separation system was applied for ns bola

sophorolipids, which are typically highly soluble at >500 g l−1, to selectively remove cells,

with a second membrane used to separate the product, the permeate also containing some

glucose, salts, and solubilised oil. This process enabled an increase in productivity from

0.37 g l−1 h−1 to 0.63 g l−1 h−1 [11]. Membrane separation with acid type sophorolipids has also

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been shown to double the productivity [11]. Membrane separation of surfactin enabled

surfactin retention in a product collection vessel whilst water and other constituents were

collected in a permeate, increasing the product concentration [22]. Combining this with cell

recycle enabled doubling of productivity to a total of around 110 mg l−1 h−1 and increased the

total surfactin output to 10 g from 3 l of broth [22].

3.3. Gravity separation

Sophorolipids can form a second phase at fermentation conditions, which can range from a

crystalline solid phase to an oily viscous phase containing sophorolipids and water,

depending on process conditions. There are reports of phase separation occurring for MELs,

cellobioselipids, polyol esters of fatty acids, and other biosurfactants [44-46].

Initial demonstrations of integrated separation for sophorolipids used a pipette to recover

sophorolipids from a shake flask [47] or turned off aeration and agitation to the bioreactor

and pumped out the sophorolipids from a sampling port [48]. These studies demonstrated

both the feasibility of recovering the sophorolipid phase, that the cells were not adversely

affected by a period of 15 minutes to 1 hour without oxygenation, and that productivity and

titer could be increased significantly by the use of this technique, reaching 387 g l−1 in 168 h,

a significant improvement on previous performance [48].

A number of studies have further developed these gravity separation techniques. Palme et al

(2010) [49] developed an ultrasound cell separation strategy that enabled them to recover

sophorolipids after biomass was removed, but had minimal success integrating it with a

fermentation broth as the ultrasound method of cell separation required a suboptimal feeding

strategy (minimal vegetable oil) for cell separation to be effective. This is because the

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presence of a second phase generally makes cell separation very difficult, particularly if the

additional phase is highly viscous. Two studies been conducted in which oil was added to the

fermentation broth in order to reach an oil concentration in the sophorolipid product of

around 10% w/w, increasing the rate of phase separation. Whilst this can give an effective

separation of a mixed oil and sophorolipid phase from the broth, the high oil concentration in

the biosurfactant product phase leads to a lower quality final product as it is difficult to

subsequently remove this oil. Further, this technique is unnecessary for gravity separation if

fermentation conditions are properly controlled [24, 50, 51].

There have been two key developments in the use of gravity separation for sophorolipid

fermentations; a bioreactor designed to separate the product by gravity and a recirculating

gravity separation system [24, 51]. To collect sophorolipids inside the fermentation vessel, a

bottom cone bioreactor section was introduced, with a sieve plate between this and the main

bioreactor. Aeration is provided both in the cone section and in the main, cylindrical,

bioreactor section. When aeration is switched off in the cone section, the sieve plate limits

turbulence in this section, and leads to product coalescence and settling; this product can then

be pumped out and collected. This resulted in an increased titer up to 477 g l−1 sophorolipid,

but at a lower productivity of 1.6 g l−1 h−1 [51].

Dolman et al (2017) [24] developed an integrated gravity separation column, recirculating the

broth in a loop from the bioreactor to the separator, where a sophorolipid rich product phase

accumulates, and then pumping the accumulated sophorolipid product from an outlet on the

separator. The separation column is designed to allow for recovery of the product from either

the broth surface or the bottom of the vessel, depending on the relative density difference

between the sophorolipid product and the fermentation broth. Further process developments

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enabled the system to reach a productivity of 5.7 g l−1 h−1, and a final titer 928 g l−1

sophorolipid product. The system has subsequently been scaled up 150 l in collaboration with

industrial surfactant producers, Croda and Allied Carbon solutions, and a spin-out company,

Holiferm, formed to commercialise the technology [52, 53, 54].

As far as the authors are aware, there are no reports of integrated production and gravity

separation for any biosurfactants other than sophorolipids; The authors recirculating gravity

separation technique has, however, also been demonstrated with MELs.

4. Scale up potential

The scale up of integrated separation systems is more complex than scale up of downstream

separation processes, primarily because negative effects on the cell biomass and hence

productivity must be avoided when integrating production and separation. As membrane

separation typically uses a first microfiltration membrane to recycle cells to the bioreactor,

these challenges are eliminated from the scale up process. Numerous filtration systems are in

commercial use for cell recycle, making this a relatively mature, albeit expensive, technology

for integrated separation, hence such scale up of membrane systems is therefore not discussed

further. Without the use of foam prevention techniques, highly foaming biosurfactants will

form a surfactant rich foam, and for other biosurfactants, a separate, viscous phase is formed.

Both a foam and this viscous phase would be challenging to filter to remove cells, making a

separation without a prior cell separation an easier as well as cheaper option.

For industrial application, the choice between a novel bioreactor configuration with

integrated separation, or a recirculating loop to transport broth to and from a separation

system is important. For foam fractionation, there is a compromise between the increased

control of the foam fractionation column and increased recovery afforded by a separate

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column, and the prevention of uncontrolled overflow afforded by an ‘in bioreactor’ column.

Maintaining oxygen transfer to the cells does not pose a major problem in either scenario, as

aeration in the foam fractionation column also acts to prevent dissolved oxygen limitation. In

any case, further process improvement is required for the use of these systems industrially to

improve titer and productivity before significant commercial use is possible.

Systems with potential for the addition of integrated gravity separation, particularly for the

production of wild type sophorolipids, have already been commercialised by a number of

surfactant manufacturers [55, 56]. Maintaining the dissolved oxygen level is key in gravity

separation systems for biosurfactants, as the laminar flow conditions necessary for separation

are incompatible with effective oxygen transfer, which requires high agitation rates and flow

in the turbulent regime. At laboratory (5l) scale, it is possible to turn off aeration and

agitation to a section of the bioreactor, or even the whole bioreactor, to enable the formation

and recovery of a separate sophorolipid phase, driven by gravity settling [57]. As the size

(height) of the bioreactor increases the settling distance also increases, resulting in longer

separation times. In industrial scale fermentations separation can take a period of several

days, limiting overall productivity by increasing the total process time, meaning fewer

production runs can be scheduled in a given campaign. When the period of time without

oxygenation reaches a critical point, thought to be in excess of 30 minutes, reduced

productivity and even cell death may be encountered [47]. It may be possible to limit the

volume of the separation segment in the bioreactor as bioreactor size increases, and so

prevent an excessive residence time increase, but it is challenging to achieve a sufficient

separation rate in order to maintain a low separation residence time.

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When using a recirculating loop separation system, residence time and separation rate

challenges are easier to overcome. There is no turbulent flow in the separator, and more

predictable transfer of fluid into and out of the unoxygenated separation zone makes it

possible to understand the impact of the integrated separation process on cell biomass.

It has been demonstrated by Dolman et al (2018) [52] that a separator could continuously

recover the sophorolipid produced in a 2 m3 bioreactor with a residence time of around 5.5

minutes; significantly below the 30 minute critical time without oxygenation, meaning that

significant negative effects, such as reduction in productivity, are avoided [52] [47]. It is also

possible to combine separation vessels in parallel if necessary, to limit the residence time in a

given separation vessel.

Another key metric to consider for scale up is the capital and developmental expenditure

required. Neither gravity-based separation set up has high intrinsic complexity, meaning

developmental costs are likely to be fairly low. Equipment costs are likely to be lower for a

recirculating gravity separating system than a new bioreactor type, but these costs could be

reduced in line with those of similar existing bioreactor units if these integrated processes

were to become mainstream.

In many cases, a single plant will produce a number of products, and existing bioreactor

capacity is available. The capability to easily retrofit an integrated separation system is

therefore important, and a recirculating loop integrated gravity separation system could easily

be retrofitted, particularly given the small footprint of the system, at less than 1/30 the size of

the associated fermentation vessel as demonstrated up to pilot scale in industrial facilities

[52].

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5. Process economics

Production costs for biosurfactants are generally reported as being primarily dependent on

bioreactor volume (influenced by productivity and somewhat by titer due to batch scheduling

and startup/shutdown time), raw material costs (influenced by choice of raw materials and

yield) and separation costs [11, 27, 37]. Productivity is most important at relatively small

scales, in the range of the current operating scales for biosurfactant production [11]. The

effects of various integrated separation technologies on process economics for several

biosurfactants are summarised in Table 2.

The bioreactor volume required for biosurfactant production is inversely proportional to the

volumetric productivity, if downtime between batches is ignored. Bioreactor capital costs are

typically assumed to be proportional to bioreactor volume within a given range, and therefore

if economies of scale are ignored, bioreactor capital costs are inversely proportional to

productivity, in other words for a bioprocess with a higher productivity a smaller, and hence

lower capital cost, bioreactor is required for a given total production. Whilst steps towards

high productivity fermentations have been made, bioreactor capital costs remain a major

economic barrier to the wider use of biosurfactants. It has been reported that a productivity of

2 g l−1 h−1 represents a threshold for commercial production of a biochemical at commodity

scale [58]. This fits with the increasing commercialisation of sophorolipids, which are

produced at productivities around 2-2.5 g l−1 h−1 without integrated separation, and the slow

pace of adoption of other biosurfactants which are made at significantly lower productivities.

At industrial scale another key cost is the startup, cleaning and sterilization costs incurred

when running sequenced batch/fed-batch production campaigns, which are often overlooked

but have been identified by industrial partners as a key target for cost reduction. Downtime

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between batches, manpower for inoculum preparation and fermentation start up, as well as

the energy and chemical costs for cleaning and sterilization all have a significant negative

effect on process economics. For a given quantity of biosurfactant, the frequency of

production campaigns is inversely proportional to the product titer. The frequency of

production campaigns dictates the frequency of startup, cleaning and sterilization, which have

a significant negative impact on overall economics, making the product titer an important

economic metric. In other words being able to run a fermentation process for longer whilst

maintaining productivity and reaching higher titers (fewer production campaigns) will reduce

production costs.

For certain systems, integrated separation has been shown to improve on all of these metrics.

For surfactin the application of cell recycle and membrane separation doubled the

productivity, but reduced titer, which the authors attribute to the recycling of toxic

compounds to the fermentation [22]. Application of foam fractionation increased the

productivity of a surfactin fermentation from 0.008 g l−1 h−1 to 0.044 g l−1 h−1 [21]. This

involved the application of a fed batch methodology rather than batch as well as the

integrated separation system, and the authors of this review consider it likely the

improvements were due to strategies other than the integrated product separation as limiting

product concentration was not reached during these fermentations.

Using integrated foam fractionation reduced the production of hydrophobin proteins but did

give the advantage of not requiring antifoam which is difficult to separate from the product

[38]. The fermentation processes for surfactin and hydrophobin proteins are in general poorly

understood, and production yields are orders of magnitude below those required for large

scale commercial production. General improvements in the fermentation process, including

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increasing cell density and converting to fed batch processes, are therefore required before

integrated separation becomes useful for removing issues caused by product accumulation,

and for the processes to become commercially viable.

Rhamnolipid producing fermentations have been developed to the extent that commercially

feasible production processes are available; but the studies that have applied foam

fractionation used fermentation processes with productivity and titer far below the state of the

art, making an analysis of the economic impact of integrated separation difficult [40, 59].

For sophorolipid producing fermentations, integrated separation has been shown by several

authors to increase product titer significantly above that possible without integrated

separation [28, 51]. Removing sophorolipid from the fermentation broth as it is produced

reduces or eliminates oxygen mass transfer limitation and alleviates the difficulties caused by

poor mixing [24]. This enables high productivity fermentations to continue past the point at

which they would normally have to be stopped, increasing the product titer and therefore

reducing start up and cleaning costs, as more sophorolipid is produced per production run,

and so fewer batches are required. Increasing the product titer also increases the fraction of

time the cells are producing sophorolipid rather than growing, increasing the yield and

productivity and hence decreasing substrate and bioreactor volume and therefore bioreactor

costs for a given quantity of sophorolipid. These and other advantages have enabled

productivity to be roughly doubled compared to the state of the art without integrated

separation [52].

At the current commercial production scales for biosurfactant production, productivity has

been reported as the most important variable [11]. The authors used the model developed by

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Ashby et al (2013) [60] to estimate the overall change in sophorolipid production costs using

the integrated separation system developed by [24]. The results show a total cost of

€1820/tonne for sophorolipid produced with integrated gravity separation calculated

compared to €5300/tonne for the state of the art without integrated separation, both at a

production scale of 10 k tonne/annum. These cost improvements, if realized, are sufficient for

the sale of sophorolipids at similar prices to other specialty surfactants, which could enable

them to take a significant market share.

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Table 2 - Effect of integrated separation on process economics. Note compromise between productivity and titer chosen for state of the art, some prior art is higher on one metric than these articles. Titers and productivities are calculated based on the liquid volume in the bioreactor.

Biosurfactant Separation technique Scale State of the art

productivity/titer

With integrated separation

productivity/titer

Recovery/purity (comparison to

commercial product)

Reference

Sophorolipid (native)

Ultrasound+gravity Lab

1.9 g l−1 h−1 /365 g l−1 [18]

0.34  g l−1 h−1, 73.8  g l−1 11%/unknown [49]Membrane separation Lab Unknown 78-82%/unknown [22]

Gravity (bioreactor+oil

separation)Lab 1.6 g l−1 h−1, 477  g l−1 unknown/74% [51]

Gravity (bioreactor only) 2.3 g l−1 h−1, 387  g l−1 Unknown/unknown [57]

Gravity (recirculating loop) Pilot 5.7  g l−1 h−1, 928  g l−1 Up to 98%/~90%. [52]

Sophorolipid (acid/bola) Ultrafiltration Pilot 0.37 g l−1 h−1/unknown 0.63 g l−1 h−1 /unknown 65%/95% [11]

SurfactinUltrafiltration Lab 0.055 g l−1 h−1 , 3.3  g l−1 0.11 g l−1 h−1, 2.42  g l−1 50% (not

considering water) [22]

Foam fractionation Lab 0.008 g l−1 h−1, 0.4  g l−1 0.044 g l−1 h−1, 0.65 g l−1 Up to 1.2% [61]

Rhamnolipids Foam fractionation Lab 1.6 g l−1 h−1 , 70  g l−1 [59] 0.078 g l−1 h−1 , 6.2  g l−1 <10% [62]Hydrophobin

proteins Foam fractionation Lab 0.004 g l-1 h-1, 0.28 g l-1 0.002 g l-1 h-1, 0.14 g l-1 <0.5% [38]

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6. ConclusionsA strong industry demand for lower cost biosurfactants is driving substantial academic and

industrial research towards integrating production and separation for biosurfactant products.

Foam formation resulting in uncontrolled overflow, viscous product accumulation limiting

oxygen mass transfer and filling of the bioreactor due to high feeding requirements are

currently limiting the economics of biosurfactant production. A number of techniques, in

particular foam fractionation and gravity separation, have been shown to be effective at

recovering the biosurfactant product, and membrane separation, gravity separation and foam

fractionation have been shown to dramatically improve process economics, doubling the

achievable product titer and bioprocess productivity. Application of these technologies in

commercial production and at scale will enable the reduced cost production of biosurfactants,

and therefore a massively expanded biosurfactant market.

Acknowledgements

The authors are grateful to the BBSRC/EPSRC NIBBs BioProNET and FoodWasteNET as

well as an EPSRC DTG PhD studentship for providing funding that supported the earlier

experimental work that underpins this manuscript. In addition, the authors would like to

thank Croda, Allied Carbon Solutions and Peel Pioneers for their support with some of the

technical work underpinning this review.

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Figure 2 - General process overview for microbial biosurfactants.

Figure 3 - Sophorolipid product accumulation and bioreactor heterogeneity during

fermentation.

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Tables

Table 1- Techniques for separating biosurfactants and their potential for use in integrated

production and separation systems, adapted from [37].

Table 2 - Effect of integrated separation on process economics. Note compromise between

productivity and titer chosen for state of the art, some prior art is higher on one metric than

these articles. Titers and productivities are calculated based on the liquid volume in the

bioreactor.

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