Integrated production and separation of biosurfactants

Process Biochemistry 83 (2019) 1–8

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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio

Review

Integrated production and separation of biosurfactants Ben M. Dolman a b c

a,b,⁎

c

, Fuju Wang , James B. Winterburn

T

a,b

School of Chemical Engineering and Analytical Science, The Mill, The University of Manchester, Manchester, M13 9PL, UK Holiferm Limited, Greenheys Building, 61 Pencroft Way, Manchester M15 6AY, UK Beijing Global Biologicals Co., Ltd No.99 Yuexiu Road Haidian District, Beijing 100096, China

A R T I C LE I N FO

A B S T R A C T

Chemical compounds studied in this article: Sophorolipid (PubChem CID: 11856871) Surfactin (PubChem CID: 443592) Rhamnolipid (PubChem CID: 5458394) Mannosylerithritol lipid

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

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 Fig. 1. 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 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

Corresponding author at: Holiferm Limited, Greenheys Building, 61 Pencroft Way, Manchester M15 6AY, UK. E-mail address: [email protected] (B.M. Dolman).

https://doi.org/10.1016/j.procbio.2019.05.002 Received 26 February 2019; Received in revised form 25 April 2019; Accepted 1 May 2019 Available online 02 May 2019 1359-5113/ © 2019 Elsevier Ltd. All rights reserved.

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

Fig. 1. Annual patents related to sophorolipid production and application, 2000–2016.

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.

2.2. 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 100 s 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 Fig. 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

2. Challenges in extracellular biosurfactant production A brief overview of a generic extracellular biosurfactant production process is shown in Fig. 2. Typically, a cell growth period with excess nitrogen source as well as carbon source is 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 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.

Fig. 2. General process overview for microbial biosurfactants.

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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 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.

Fig. 3. Sophorolipid product accumulation and bioreactor heterogeneity during fermentation.

challenges. Attempts to reduce the effects of product accumulation and viscosity have included preventing product expression during the cell growth phase [27], as well as integrated product recovery which is discussed in Section 3. 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 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].

3.1. 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

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

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 Any biosurfactant Lipopeptides, glycolipids

Minimal (with solvents used), cell death

Adsorption Membranes/filtration Foam fractionation Gravity Gravity Gravity Gravity

separation separation separation separation

(no salt) (salting out) (acid precipitation) (crystallisation)

Rhamnolipids, lipopeptides, sophorolipids, MELs Sophorolipids, MELs Sophorolipids, MELs, rhamnolipids Surfactin, rhamnolipid Sophorolipids, cellobioselipids

Potential to use with a cell separation step before adsorption, cells would foul column. Successfully demonstrated, but potential for use with dissolved biosurfactants. Precipitated biosurfactants may cause fouling. Successfully demonstrated Successfully demonstrated. Difficult to apply without cell damage Difficult to apply without cell damage. Proof of principle verified.

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reasonable cost.

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 with a dilute product, concentrating products from initial broth concentrations in the order of 10 s mg l−1 to final foamate concentrations of 100 s 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]. 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

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 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 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 min to 1 h 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 4

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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 (5 l) 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 min, 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. 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 min; significantly below the 30 min 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].

a suboptimal feeding strategy (minimal vegetable oil) for cell separation to be effective. This is because the 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 enabled the system to reach a productivity of 5.7 g l−1 h−1, and a final titer 928g 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 [52–54]. 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 column, and the prevention of uncontrolled overflow afforded by an ‘in bioreactor’ column. Maintaining oxygen transfer to the cells

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 5

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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 Membrane separation Gravity (bioreactor + oil separation) Gravity (bioreactor only) Gravity (recirculating loop) Ultrafiltration

Lab Lab 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 Unknown 1.6 g l−1 h−1, 477 g l−1

11%/unknown 78-82%/unknown unknown/74%

[49] [22] [51]

Pilot Pilot

0.37 g l−1 h−1/unknown

2.3 g l−1 h−1, 387 g l−1 5.7 g l−1 h−1, 928 g l−1 0.63 g l−1 h−1 /unknown

Unknown/unknown Up to 98%/˜90%. 65%/95%

[57] [52] [11]

Ultrafiltration Foam fractionation Foam fractionation Foam fractionation

Lab Lab Lab Lab

0.055 g l−1 h−1, 3.3 g l−1 0.008 g l−1 h−1, 0.4 g l−1 1.6 g l−1 h−1, 70 g l−1 [59] 0.004 g l−1 h−1, 0.28 g l−1

0.11 g l−1 h−1, 2.42 g l−1 0.044 g l−1 h−1, 0.65 g l−1 0.078 g l−1 h−1, 6.2 g l−1 0.002 g l−1 h−1, 0.14 g l−1

50% (not considering water) Up to 1.2% < 10% < 0.5%

[22] [61] [62] [38]

Sophorolipid (acid/ bola) Surfactin Rhamnolipids Hydrophobin proteins

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 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 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.

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 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

6. Conclusions A 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 6

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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.

[23]

[24] [25]

[26] [27]

Acknowledgements

[28]

The authors are grateful to the Biotechnology and Biosciences Research Council and Engineering and Physical Sciences Research Council Networks in Industrial Biotechnology, (BBSRC/EPSRC NIBBs) BioProNET and FoodWasteNET as well as an Engineering and Physical Sciences (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.

[29] [30] [31] [32]

[33]

References

[34]

[1] A. Kumar, Global market for surfactant chemicals and materials, (2018). [2] Ra. Markets, Global surfactant market, companies profiles, size, share, growth, Trends and Forecast to 2025, (2018). [3] J. Geier, W. Uter, C. Pirker, P.J. Frosch, Patch testing with the irritant sodium lauryl sulfate (SLS) is useful in interpreting weak reactions to contact allergens as allergic or irritant, Contact Derm. 48 (2003) 99–107. [4] S. Asplund, Sustainable bio-based surfactants for everyone, (2019). [5] I.N.A. Van Bogaert, K. Saerens, C. De Muynck, D. Develter, W. Soetaert, E.J. Vandamme, Microbial production and application of sophorolipids, Appl. Microbiol. Biotechnol. 76 (2007) 23–34. [6] D.W.G. Develter, L.M.L. Lauryssen, Properties and industrial applications of sophorolipids, Eur. J. Lipid Sci. Technol. 112 (2010) 628–638. [7] A. Franzetti, E. Tamburini, I.M. Banat, Applications of biological surface active compounds in remediation technologies, Biosurfactants 672 (2010) 121–134. [8] N. Lourith, M. Kanlayavattanakul, Natural surfactants used in cosmetics: glycolipids, Int. J. Cosmet. Sci. 31 (2009) 255–261. [9] E.J. Gudiña, V. Rangarajan, R. Sen, L.R. Rodrigues, Potential therapeutic applications of biosurfactants, Trends Pharmacol. Sci. 34 (2013) 667–675. [10] L. Rodrigues, I.M. Banat, J. Teixeira, R. Oliveira, Biosurfactants: potential applications in medicine, J. Antimicrob. Chemother. 57 (2006) 609–618. [11] S. Roelants, L. Van Renterghem, K. Maes, B. Everaert, B. Vanlerberghe, S. Demaeseneire, W. Soetaert, Microbial biosurfactants: from lab to market, in: C. Press (Ed.), Microbial Biosurfactants and Their Environmental and Industrial Applications, 2019. [12] P.B. Singh, H.S. Saini, Exploitation of agro-industrial wastes to produce low-cost microbial surfactants, Biotransformation of Waste Biomass into High Value Biochemicals (2014) 445–471. [13] I.N.A. Van Bogaert, J. Zhang, W. Soetaert, Microbial synthesis of sophorolipids, Process. Biochem. 46 (2011) 821–833. [14] I.M. Banat, S.K. Satpute, S.S. Cameotra, R. Patil, N.V. Nyayanit, Cost effective technologies and renewable substrates for biosurfactants’ production, Front. Microbiol. 5 (2014). [15] L. Van Renterghem, Roelants SLKW, N. Baccile, K. Uyttersprot, M.C. Taelman, B. Everaert, S. Mincke, S. Ledegen, S. Debrouwer, K. Scholtens, C. Stevens, W. Soetaert, From lab to market: an integrated bioprocess design approach for newto-nature biosurfactants produced by Starmerella bombicola, Biotechnol. Bioeng. 115 (2018) 1195–1206. [16] L. Zhu, X. Yang, C. Xue, Y. Chen, L. Qu, W. Lu, Enhanced rhamnolipids production by Pseudomonas aeruginosa based on a pH stage-controlled fed-batch fermentation process, Bioresour. Technol. 117 (2012) 208–213. [17] J.A. Casas, F. García-Ochoa, Sophorolipid production by Candida bombicola: medium composition and culture methods, J. Biosci. Bioeng. 88 (1999) 488–494. [18] Y.-B. Kim, H.S. Yun, E.-K. Kim, Enhanced sophorolipid production by feeding-ratecontrolled fed-batch culture, Bioresour. Technol. 100 (2009) 6028–6032. [19] U. Rau, L.A. Nguyen, H. Roeper, H. Koch, S. Lang, Fed-batch bioreactor production of mannosylerythritol lipids secreted by Pseudozyma aphidis, Appl. Microbiol. Biotechnol. 68 (2005) 607–613. [20] J.B. Winterburn, P.J. Martin, Foam mitigation and exploitation in biosurfactant production, Biotechnol. Lett. 34 (2012) 187–195. [21] C. Kaisermann, Bioprocess Intensification of Surfactin Production, The University of Manchester, 2017. [22] F. Coutte, D. Lecouturier, V. Leclère, M. Béchet, P. Jacques, P. Dhulster, New integrated bioprocess for the continuous production, extraction and purification of

[35]

[36] [37] [38]

[39] [40]

[41] [42] [43]

[44] [45]

[46]

[47]

[48] [49]

[50]

[51]

[52]

[53] [54] [55] [56]

7

lipopeptides produced by Bacillus subtilis in membrane bioreactor, Process. Biochem. 48 (2013) 25–32. A.-M. Davila, R. Marchal, J.-P. Vandecasteele, Kinetics and balance of a fermentation free from product inhibition: sophorose lipid production by Candida bombicola, Appl. Microbiol. Biotechnol. 38 (1992) 6–11. B.M. Dolman, C. Kaisermann, P.J. Martin, J.B. Winterburn, Integrated sophorolipid production and gravity separation, Process. Biochem. 54 (2017) 162–171. V.K. Morya, J.H. Park, T.J. Kim, S. Jeon, E.K. Kim, Production and characterization of low molecular weight sophorolipid under fed-batch culture, Bioresour. Technol. 143 (2013) 282–288. S. Bages-Estopa, D.A. White, J.B. Winterburn, C. Webb, P.J. Martin, Production and separation of a trehalolipid biosurfactant, Biochem. Eng. J. 139 (2018) 85–94. R. Gao, M. Falkeborg, X. Xu, Z. Guo, Production of sophorolipids with enhanced volumetric productivity by means of high cell density fermentation, Appl. Microbiol. Biotechnol. 97 (2013) 1103–1111. B. Dolman, J. Winterburn, Separating lipids from a fermentation broth can dramatically improve bioprocess performance, Int. News Fats, Oils Relat. Mater. 28 (2017) 24–26. U. Rau, S. Hammen, R. Heckmann, V. Wray, S. Lang, Sophorolipids: a source for novel compounds, Ind. Crops Prod. 13 (2001) 85–92. D. Stark, U. von Stockar, In situ product removal (ISPR) in whole cell biotechnology during the last twenty years, Adv. Biochem. Eng. Biotechnol. 80 (2003) 149–175. M.C. Cuellar, A.J.J. Straathof, CHAPTER 4: improving fermentation by product removal, RSC Green Chemistry vol. 2018-January, (2018), pp. 86–108. J.B. Winterburn, A.B. Russell, P.J. Martin, Integrated recirculating foam fractionation for the continuous recovery of biosurfactant from fermenters, Biochem. Eng. J. 54 (2011) 132–139. N. Pandurić, A. Šalić, B. Zelić, Fully integrated biotransformation of fumaric acid by permeabilized baker’s yeast cells with in situ separation of L-malic acid using ultrafiltration, acidification and electrodialysis, Biochem. Eng. J. 125 (2017) 221–229. P.L. Chew, M.S.M. Annuar, P.L. Show, T.C. Ling, Extractive bioconversion of poly-εcaprolactone by Burkholderia cepacia lipase in an aqueous two-phase system, Biochem. Eng. J. 101 (2015) 9–17. W. Van Hecke, G. Kaur, H. De Wever, Advances in in-situ product recovery (ISPR) in whole cell biotechnology during the last decade, Biotechnol. Adv. 32 (2014) 1245–1255. N. Kosaric, V.S. Fazilet, Biosurfactants:Production and Utilization-Processes, Technologies and Economics, CRC Press, 2014. T.J.P. Smyth, A. Perfumo, R. Marchant, I.M. Banat, Isolation and Analysis of Low Molecular Weight Microbial Glycolipids. (2010), pp. 3705–3723. J.B. Winterburn, A.B. Russell, P.J. Martin, Characterisation of HFBII biosurfactant production and foam fractionation with and without antifoaming agents, Appl. Microbiol. Biotechnol. 90 (2011) 911–920. C.-Y. Chen, S.C. Baker, R.C. Darton, Batch production of biosurfactant with foam fractionation, J. Chem. Technol. Biotechnol. 81 (2006) 1923–1931. J. Beuker, A. Steier, A. Wittgens, F. Rosenau, M. Henkel, R. Hausmann, Integrated foam fractionation for heterologous rhamnolipid production with recombinant Pseudomonas putida in a bioreactor, AMB Express 6 (2016) 11. In situ separation of amphipathic compound by foam adsorption. Bio-surfactant production and separation device. CN203545999U. S. Alonso, P.J. Martin, Impact of foaming on surfactin production by Bacillus subtilis: implications on the development of integrated in situ foam fractionation removal systems, Biochem. Eng. J. 110 (2016) 125–133. B. Lemmens, Novel Production Strategies for Biosurfactants (2014). U. Rau, L.A. Nguyen, H. Roeper, H. Koch, S. Lang, Downstream processing of mannosylerythritol lipids produced by Pseudozyma aphidis, Eur. J. Lipid Sci. Technol. 107 (2005) 373–380. L.A. Garay, I.R. Sitepu, T. Cajka, J. Xu, H.E. Teh, J.B. German, Z. Pan, S.R. Dungan, D.E. Block, K.L. Boundy-Mills, Extracellular fungal polyol lipids: a new class of potential high value lipids, Biotechnol. Adv. 36 (2018) 397–414. V. Guilmanov, A. Ballistreri, G. Impallomeni, R.A. Gross, Oxygen transfer rate and sophorose lipid production by Candida bombicola, Biotechnol. Bioeng. 77 (2002) 489–494. Institut Francais Du Petrole. Method of production of sophorosides by fermentation with fed batch supply of fatty acid esters or oils. US5879913 A. O. Palme, G. Comanescu, I. Stoineva, S. Radel, E. Benes, D. Develter, V. Wray, S. Lang, Sophorolipids from Candida bombicola: cell separation by ultrasonic particle manipulation, Eur. J. Lipid Sci. Technol. 112 (2010) 663–673. Z. Liu, X. Tian, Y. Chen, Y. Lin, A. Mohsin, J. Chu, Efficient sophorolipids production via a novel in situ separation technology by Starmerella bombicola, Process. Biochem. (2018). Y. Zhang, D. Jia, W. Sun, X. Yang, C. Zhang, F. Zhao, W. Lu, Semicontinuous sophorolipid fermentation using a novel bioreactor with dual ventilation pipes and dual sieve-plates coupled with a novel separation system, Microb. Biotechnol. (2017). B. Dolman, S. Bages Estopa, S. Shiba, M. Panhalker, T. Kawakami, M. Yashiro, J. Winterburn, Enhancing food waste based sophorolipid production via novel separation at scale (2018). B. Dolman, J. Winterburn, Collaborative Development of Glycolipid Separation Technology to Reduce Costs, (2018). B. Dolman, Holiferm Develops and Offers Licences to Holistically Improved Fermentation Technology for Massive Economic Improvements, (2019). J. Krauter, Evonik Commercializes Biosurfactants, (2016). J. Novoseletsky, Actera Ingredients and Allied Carbon Solution Announce Collaboration, (2018).

Process Biochemistry 83 (2019) 1–8

B.M. Dolman, et al.

[59] Semi-continuous process for the production of rhamnolipids at high yield and titer. [60] R.D. Ashby, A.J. McAloon, D.K.Y. Solaiman, W.C. Yee, M. Reed, A process model for approximating the production costs of the fermentative synthesis of sophorolipids, J. Surfactants Deterg. 16 (2013) 683–691. [61] D.A. Davis, H.C. Lynch, J. Varley, The application of foaming for the recovery of Surfactin from B. Subtilis ATCC 21332 cultures, Enzyme Microb. Technol. 28 (2001) 346–354. [62] I. Anic, I. Apolonia, P. Franco, R. Wichmann, Production of rhamnolipids by integrated foam adsorption in a bioreactor system, AMB Express 8 (2018).

[57] R. Marchal, J. Lemal, C. Sulzer, A.M. Davila, Production of Sophorolipid Acetate Acids From Oils or Esters: Google Patents, (1999). [58] A.L. Meadows, K.M. Hawkins, Y. Tsegaye, E. Antipov, Y. Kim, L. Raetz, R.H. Dahl, A. Tai, T. Mahatdejkul-Meadows, L. Xu, L. Zhao, M.S. Dasika, A. Murarka, J. Lenihan, D. Eng, J.S. Leng, C.-L. Liu, J.W. Wenger, H. Jiang, L. Chao, P. Westfall, J. Lai, S. Ganesan, P. Jackson, R. Mans, D. Platt, C.D. Reeves, P.R. Saija, G. Wichmann, V.F. Holmes, K. Benjamin, P.W. Hill, T.S. Gardner, A.E. Tsong, Rewriting yeast central carbon metabolism for industrial isoprenoid production, Nature 537 (2016) 694.

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