Review

Microbial biosurfactants: challenges and opportunities for future exploitation

Roger Marchant and Ibrahim M. Banat
School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, UK

The drive for industrial sustainability has pushed biosurfactants to the top of the agenda of many companies. Biosurfactants offer the possibility of replacing chemical surfactants, produced from nonrenewable resources, with alternatives produced from cheap renewable feedstocks. Biosurfactants are also attractive because they are less damaging to the environment yet are robust enough for industrial use. The most promising biosurfactants at the present time are the glycolipids, sophorolipids produced by Candida yeasts, mannosylerythritol lipids (MELs) produced by Pseudozyma yeasts, and rhamnolipids produced by Pseudomonas. Despite the current enthusiasm for these compounds several residual problems remain. This review highlights remaining problems and indicates the prospects for imminent commercial exploitation of a new generation of microbial biosurfactants. The move towards biosurfactants Chemical surfactants have a major impact on all our lives because they comprise a major component of many of the everyday products we use. These chemical surfactants, many of which are alkyl sulfates or sulfonates with straight or branched chains and come from either petrochemical or oleochemical sources [1], can be found as components of laundry products, surface cleaning agents, concrete additives, cosmetics, and pharmaceuticals, used in agro food processing and used in the petroleum industry. The worldwide use of surfactants has grown enormously over the past few decades, although exact gures for production are difcult to determine in such a mixed market. However, quantities of approximately 9 million tonnes in 1995 rising to 13 million tonnes in 2008 are probably reasonable estimates [2]. It has also been estimated that in the EU, 50% of the surfactants produced have hydrophobic tails derived from palm or coconut oil [2]. The major shift in attitude towards surfactants that has occurred in the past few years has been driven by the sustainability agenda. Companies using surfactants in their products are now looking to replace some or all of the chemical surfactants with sustainable biosurfactants, that is, surfactant molecules produced principally by microorganisms from sustainable feedstocks. These molecules have the added advantage that, although they are stable at relatively high
Corresponding author: Marchant, R. (r.marchant@ulster.ac.uk). Keywords: rhamnolipids; sophorolipids; mannosylerythritol lipids; MEOR; biofilms.

temperature and in adverse environments, they are still readily biodegradable in the environment if, or when, discharged. A few commercial products, mainly from the far east in Asia, have already included biosurfactants in their formulations, however, several problems remain before more widespread use can be envisaged. These problems relate to yield and cost of production, including downstream processing, but also to the tailoring of the molecules to specic applications. Surfactant molecules are described as amphiphilic, that is, they have a hydrophilic end and a hydrophobic end, which allows them to interact at the interfaces between aqueous and nonaqueous systems, including air. Their effects in these systems include the reduction of surface tension, emulsication, wetting, and foaming and depend on the exact structure of the individual molecules (Box 1). Microbially produced biosurfactants can be broadly classied into low molecular weight (glycolipids, lipopeptides, and avolipids) [3] and high molecular weight molecules (polysaccharides, proteins, lipopolysaccharides, and lipoproteins) [4]. Of these different forms, the low molecular weight glycolipids are perhaps the most interesting for exploitation in the near future, and it is these that this review will focus on. In this review, the current state of knowledge about these molecules will be surveyed, and remaining problems concerning exploitation and production will be highlighted. With this information, the reader will be able to make a judgement about how imminent is the widespread incorporation of microbial biosurfactants in commercial products. Cleaning applications One of the major domestic product applications of biosurfactants is in the area of laundry products. At present, the surfactant content of the liquids and powders manufactured is largely alkyl sulfonates such as linear alkylbenzene sulfonates (LASs). However, the glycolipid biosurfactants, sophorolipid produced by yeasts of the genus Candida, rhamnolipids produced by Pseudomonas aeruginosa, and MELs produced by basidiomycetous yeasts of the genus Pseudozyma and the fungus Ustilago are possible candidates to be used as, at least, partial replacements for LAS [5]. One of the major challenges in the use of these biosurfactants is that each organism produces a mixture of congener molecules with a range of different structures and therefore properties. In the case

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0167-7799/$ see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2012.07.003 Trends in Biotechnology, November 2012, Vol. 30, No. 11

Review
Box 1. Surfactants
The term surfactant was derived from the phrase surface active agents and describes the activity of these amphiphilic molecules at the interfaces between different phases, gas, liquid, and solid. Surfactants are able to act as detergents, wetting agents, emulsifiers, dispersants, and foaming agents, and form major ingredients of many product formulations ranging from household detergents, shampoos, personal care products, and pharmaceuticals to paints. The worldwide use of surfactants is enormous, estimated in 2008 to be 13 million tonnes per annum (p.a.) [2], with a predicted increase in use of approximately 2% p.a., and currently focuses on chemical surfactants, principally LASs and alkyl phenol ethoxylates (APEs). In an aqueous environment, surfactants form aggregate structures called micelles in which the hydrophobic tails of the molecules are protected from contact with water. Depending on the molecular architecture of the surfactant, these micelles may be spherical, wormlike, or lamellar sheets or adopt other topologies. The aggregates form to minimise free energy of the solution and are therefore dynamic and highly dependent on the physical conditions such as temperature [1]. The critical micelle concentration (CMC) is defined as the concentration above which micelles are formed; this value is strongly dependent on temperature, pressure, and the presence of other electrolytes. Below the CMC, surface tension (ST) in aqueous systems falls from a maximum value of 72 mN/m for pure water to a minimum possible value of approximately 29 mN/m [1]. Once the CMC is reached, ST remains more or less constant. ST and interfacial tension (IT) between liquid phases are useful measures to determine whether a microbial culture is producing biosurfactant, but cannot be used in a quantitative manner, because once the minimum ST or IT is reached, further production of biosurfactant does not lead to any change in value. The different congeners in a biosurfactant mixture, produced by a single organism, show different micellar topologies and therefore behave differently when used in product formulations [7]. It is this fact that is driving the search for designer biosurfactants and for ways of producing single biosurfactant molecules rather than mixtures. The behaviour of biosurfactants in solution and at surfaces can be investigated using techniques such as small-angle neutron scattering (SANS) [6,7,9,10], although this requires major equipment facilities.

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(a)
1 2 3 4 5 6 7 8 9

Congener structure

% Abundance

(b)
CH2OR2 OH HO OH O HO

CH2OR1 OH O O

CH3 CH

Acidic, C18:1 Acidic, C18:1, 1Ac Acidic, C18:2, 2Ac Acidic, C18:1, 2Ac Acidic, C18:0, 2Ac Lactonic, C18:1, 1Ac Lactonic, C18:2, 2Ac Lactonic, C18:2, 2Ac Lactonic, C16:0, 2Ac

6.5 4.9 2.8 48.1 2.8

O (CH2)n

COOH 3.06 2.7 CH2OR1 2.2 1.1 4.6 10.0 OH 12 Lactonic, C18:0, 2Ac 4.1 O C O

(c)
CH2OR2 OH O HO

CH3 O CH

OH

10 Lactonic, C18:1, 2Ac 11 Lactonic, C18:1, 2Ac

O (CH2)n

TRENDS in Biotechnology

Figure 1. (a) Representative chemical composition of sophorolipid mixture produced by Candida apicola ATCC 96134 in a bioreactor fermentation with oleic acid as the major carbon source based on HPLC data. Chemical structures for the (b) acidic and (c) lactonic forms of sophorolipid. From these structures, it is clear why the different congeners of the biosurfactants behave differently during selfassembly in solution and also interact differently at surfaces.

of sophorolipids, although the alkyl chain length is consistent, the degree of unsaturation is not and the number of acyl groups varies from none to two, with two major congurations of the molecular structure, that is, acidic and lactonic (Figure 1). It is possible to isolate and separate the various congeners, including the acidic and lactonic forms [3], however, on a commercial scale, such downstream processing would be unlikely to be economic. In order to understand the behaviour of the different sophorolipid molecules, neutron beam scattering has been used to investigate the self-assembly and surface activity of the molecules alone and in combination with chemical surfactants [6,7]. Although the neutron beam scattering technique can be applied to the molecules in the natural state, their investigation is greatly aided if the molecules can be labelled with deuterium. This can be achieved selectively through the use of D2O and deuterium-labelled substrates in the growth medium of the Candida spp. [8]. Interestingly, the yeasts were largely unaffected by the presence of deuterium in the medium, in marked contrast to the bacteria also used, which required extensive adaptation. Sophorolipids produced by Candida bombicola have already been incorporated in some domestic products produced in Korea. Another major candidate to be considered for use in this eld is the rhamnolipids produced by P. aeruginosa. Once

again, several different molecules are produced by this bacterium, with differing alkyl chain lengths ranging from 8 to 12 carbon atoms, although two major molecules are produced, the mono-rhamnolipid with two C10 alkyl chains and the di-rhamnolipid also with two C10 alkyl chains (Figure 2). Chromatographic separation of the congeners is possible but again not economic on a large scale, although this methodology has been used to investigate the behaviour of rhamnolipids using the neutron beam scattering technique in combination with deuterium labelling [9,10]. Not surprisingly, different behaviour has been noted for the mono and di-rhamnolipids, which clearly indicates that an ability to manipulate the composition of the rhamnolipid mixture would be an advantage in commercial applications. This aspect will be dealt with further under the section on designer biosurfactants. One significant problem with the rhamnolipids until recently was the fact that they were only known to be produced by P. aeruginosa, a class II opportunistic pathogen; something that provides a disincentive for large-scale production. Recently, two nonpathogenic, related bacteria have been identied as rhamnolipid producers, although the rhamnolipids produced are different to those produced by P. aeruginosa. Pseudomonas chlororaphis produces only mono-rhamnolipid [11], whereas Burkholderia thailandensis produces predominantly di-rhamnolipid with longer alkyl chains than that produced by P. aeruginosa [12]. It is possible that the genetic characteristics of these two organisms could be exploited to produce specic rhamnolipids for particular applications. If biosurfactants are to replace chemical surfactants in laundry products, then factors such as the effects of hard water, temperature, and compatibility with microbial enzymes included in the formulations have to be considered. Temperature sensitivity has become a low priority with the drive to reduce washing temperatures as an energy saving measure.
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ST5HEXEXTRACT # 16 RT: 0.010.09 AV:6 NL:4.64E5 F: c ms[175.001000.00]
100 95 649.2

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Rha-Rha-C10-C10
OH

90 85 80 75 70 65

OH O O HO H3C O O O OH OH
H3C HO HO O

Rha-C10-C10
503.2 H3C HO HO O O

CH3 CH3

Relave abundance

60 55 50 45 40 35 30 25 20 15 10 5 0 200 250 300

Monorhamnolipid

OH

CH3

650.3

CH3
677.3 761.5 504.3 325.3 333.1 303.1 359.0 195.9 248.6 289.9 350 475.1 437.5 457.0 400 450 500 531.2 622.1 561.2 588.4 550 600 m/z 650 747.4 678.1 711.8

Dirhamnolipid
989.2 975.1 762.6 815.7 845.0 915.0 955.2

700

750

800

850

900

950

1000

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Figure 2. Mass spectroscopy data showing the range of rhamnolipid congeners produced by Pseudomonas aeruginosa strain ST5 with the two main products the mono and di-rhamno forms with two C10 alkyl chains.

The nal group of microbial glycolipids with perceived potential in this area are the MELs produced by the basidiomycetous yeasts of the genus Pseudozyma and also by the fungus Ustilago. MELs from Pseudozyma have been extensively investigated [13]. As with the other producer organisms, a range of different MEL molecules are produced, differing in alkyl chain length and degree of acylation. One major advantage of the MEL producers, like the sophorolipid producers, is that resting cells continue to synthesize the biosurfactant, allowing yields to exceed 100 g/l [14]. Biolm prevention and disruption Although much of the laboratory-based work with bacteria is conducted with planktonic cultures, mixed species biolms are a more common mode of growth for these organisms. Biolms have a complex structure that allows cell communication, (quorum sensing), to take place, which also acts as a protection for the cells from external factors such as antibiotics [15]. Biolms can develop on a wide range of surfaces including domestic household areas and medical devices such as catheters and prostheses. Biosurfactants are believed to play a major role in the development and maintenance of biolms in P. aeruginosa [16]; partly at least through the maintenance of water channels through the biolm. Attention is now turning to the possibility that biosurfactants can be used to disrupt established biolms and to prevent the development of new ones. Rhamnolipids can inhibit the adhesion of yeasts and bacteria to voice prostheses [17]
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and can mediate the disruption of established biolms [1821]. The lipopeptide surfactants putisolvin I and II produced by Pseudomonas putida are able to inhibit the formation of biolms of other Pseudomonas strains and indeed to break down established biolms [22]. Although it is useful at a preliminary stage to examine the effect of the biosurfactants alone on biolms, the next step must be to determine the interactions between biosurfactants and other components of cleaning agents such as chemical surfactants. Moreover, pH and other compounds might boost activity, as seen in the synergistic effect of pyrophosphate and sodium dodecyl sulfate (SDS) on periodontal pathogens [23]. Biocidal activity and wound healing Biosurfactants can have a strong killing action on some types of cells, with lysis of red blood cells or fungal zoospores used as a bioassay. The interesting question, however, is whether more resistant cells, for example, bacteria with cell walls, may be killed by biosurfactants. For example, sophorolipids improve sepsis survival in model systems in animals [24,25], however, in vitro, sophorolipids have no antibacterial activity [26]. At the present time, very few studies have been directed towards the possible wound healing properties of biosurfactants. Rhamnolipids have also been used in two studies [27,28], and encouraging results have been reported using low concentrations (0.1%) to treat ulcers and burns. This area of study certainly warrants further investigation and extension to

Review
other biosurfactant molecules because there would be a large market for a safe, cheap wound healing additive for over-the-counter products. Environmental applications Many different functions have been ascribed to the biosurfactants produced by microorganisms; one of which is their involvement in the metabolism of hydrophobic substrates [29]. In aqueous environments, the interfacial activity of biosurfactants and bioemulsiers can make substrates like hydrocarbons more amenable to the degradative activity of the cell. This being the case, we might expect that the majority of bacteria that utilise hydrophobic substrates would be biosurfactant producers but this is not so. We may therefore ask whether the addition of biosurfactant to the environment of a non-producer could improve the ability of that organism to degrade a hydrophobic substrate. The obvious situation where this might be advantageous would be in the eld of bioremediation, particularly in situ bioremediation. The mechanisms involved in interactions between biosurfactants or the microbial cells and immiscible hydrocarbons include: (i) emulsication; (ii) adhesion/de-adhesion of microorganisms to and from hydrocarbons; (iii) micellarisation; and (iv) desorption of contaminants; all of which are expected to enhance the rates of biodegradative bioremediation. Current literature generally supports such conclusions, however, some cases in which complex interactions among microbial cells, organic substrates, surface active compounds and their environment, leading to inhibition of biodegradation, have also been reported [30]. One group of bacteria that have been examined as potentially useful for clean-up of oil spills and contamination are the thermophilic bacilli of the genus Geobacillus [31], which do not produce any biosurfactant. These organisms seem to have great potential because they are present in seemingly all soil environments in a dormant state, having been distributed through atmospheric transport [32,33]. Simply raising the temperature of the environment allows them to become active and to compete effectively with other soil organisms [34]. In order to enhance the rate and extent of hydrocarbon degradation, inorganic nutrients and biosurfactants can be added to the system. In experiments using soil microcosms, the maximum degradation rate and extent of selected hydrocarbons was achieved when both the nutrient supplements and biosurfactant were added [35,36]. It is therefore clear that the addition of biosurfactants, even to organisms that do not produce their own, can have highly benecial effects. At the present time, marine and coastal oil spills are treated, at least in part, by the use of chemical surfactants and emulsiers, future investigation of the use of biosurfactants in their place is certainly a fruitful avenue for investigation. Biosurfactants also have extensive potential application in the petroleum industry, which in turn affects the environment (Box 2). Microbially enhanced oil recovery (MEOR) is a technique that either uses a crude preparation of biosurfactant or a whole killed culture to liberate crude oil from a binding substrate. Poor oil recovery in many existing producing wells is usually due to several factors.

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The main factor is the low permeability of some reservoirs or the high viscosity of oil, which results in poor mobility. High interfacial tensions between the water and oil may also result in high capillary forces retaining the oil in the reservoir rock. Most of the oil remains in the reservoir following primary and secondary recovery techniques, thus, interest has developed in tertiary recovery techniques [37]. A form of MEOR has been pioneered effectively at full scale to recover oil from the sludge that accumulates in oil storage tanks [38], producing a situation where the cost of carrying out the process is completely offset by the value of the recovered oil. A second potential application for biosurfactants in the oil industry is in the initial process of drilling where chemical surfactants are currently used. Techniques involving the use of chemical or physical processes such as pressurisation, water ooding, or steaming, are often inapplicable for many oil reservoirs [39]. The use of chemical surfactants for mobilising or sweeping oil reservoirs is an unfavourable practice that is hazardous, costly and leaves undesirable residues that are difcult to dispose of without adversely affecting the environment [40]. This is particularly the case in marine environments where the use of biodegradable biosurfactants rather than chemical surfactants would have major environmental benets. Designer biosurfactants Microbial biosurfactant producers invariably give a product that comprises a range of different congeners built around a basic structure. The different structures dictate the properties of the various molecules with effects on, for example, water solubility and micelle structure. Equally clearly, different applications in commercial products may require specic properties for the surfactant used. The ability to select or design specic biosurfactants is therefore highly desirable. As we can see, isolation and purication of individual components is feasible but unlikely to be economic on a large scale [3]. The next simplest approach is to modify growth and production conditions or to select specic strains of the producer organisms. In practice the mixed composition of biosurfactants produced varies only within a limited range, restricting the use of this approach. Some success has, however, been achieved with sophorolipids by using unconventional hydrophobic substrates, thus modifying the alkyl chains of the sophorolipids [41]. One simple and effective approach to biosurfactant modication used a naturally produced acylated MEL and removed the acyl groups with a lipase-catalysed hydrolysis, producing a nonacylated product (MEL-D) [42]. They are able to show a higher critical aggregation concentration and excellent surface tension, lowering capacity for the deacylated MELs, indicating that the new MEL-D may have applications in elds in which a lamellar-forming glycolipid is required. A more difcult and costly strategy is to investigate genetic modication of the producer organisms. The synthetic pathway for the P. aeruginosa rhamnolipids is a simple one consisting of two control genes RhlI and RhlR and three synthetic genes RhlA, RhlB, and RhlC. All but RhlC are located in a single operon [43]. It is thus feasible
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Box 2. MEOR
As a general rule, oil fields are developed in three stages that are typical for most reservoirs, including heavy crude oil worldwide. Stage 1, primary recovery: production under natural pressure and flow characteristics of the crude lead to up to 15% of oil in place recovered. Stage 2, secondary recovery: the oil well is flooded with water or other substances including CO2 injection, alkaline surfactant polymers (ASPs), solvents or steam to drive out an additional 1520% through sweeping the oil towards the producing wells by displacing the crude oil. Stage 3, tertiary recovery or enhanced oil recovery (EOR): remaining oil is extracted after primary and secondary recovery methods are exhausted or no longer economic. Several methods, including MEOR, have been gaining significance as a process to recover up to 10% more oil from the well. MEOR utilises microorganisms and/or their metabolic end products for recovery of residual oil that is hindered by poor oil recovery due to low permeability of some reservoirs or high viscosity resulting in poor mobility [28]. MEOR therefore results in reduction of oil viscosity through partial break down of the large molecular structure of crude oil,

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making it more fluid; production of CO2 gas as a byproduct of microbial metabolism, which both pressurises the reservoir and moves upward, displacing oil in the well; production of biomass that accumulates between the oil and the rock surface of the well, physically displacing the oil and making it easier to recover from the well; selective plugging through exopolysaccharide production that plugs large pores in the rocks forcing movement through different channels sweeping the oil out; production of biosurfactants that act as slippery detergents, helping the oil move more freely away from rocks and crevices so that it may travel more easily out of the well. MEOR and the use of biosurfactants reduce the need to use harsh chemicals during oil drilling and have several environmental advantages; they are achieved either through ex situ production and injection into oil reservoirs, or through injection of selected microorganisms to produce biosurfactants in situ, or through enhancing indigenous microbial cultures to produce such compounds [29]. This has been an area of great interest and literature debate during the past decade and large field trials are envisaged in the near future (Figure I).

Injecon well

Producon well

(Bacteria, nutrients, and/or biosurfactants)

Pressing water containing microorganisms biosurfactants nutrients Enhanced mobility Biodegradaon of crude oil (to low molecular weight)

Microbial metabolites/ biosurfactants

Crude oil

Advanced water

Gas Acid Biomass Polymer

Improvement of crude oil mobility Improvement of oil reservoir

percolaon Enhanced oil recovery

TRENDS in Biotechnology

Figure I. Diagram showing the possible use of biosurfactants for MEOR.

to contemplate cloning the pathway into another host bacterium, for example, Escherichia coli. RhlA and RhlB genes in E. coli have already been cloned and expressed a long time ago [44]. We might expect this combination to yield only mono-rhamnolipid because RhlC codes for the second rhamnosyl transferase, which converts mono- to dirhamnolipid. This was indeed the outcome but with only small yields recorded. It does seem unlikely that this
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approach can be completely successful because production of large quantities of biosurfactant depends on the metabolic ux within the bacterial cell, providing the precursors for synthesis. An alternative is to leave the genes in P. aeruginosa but to knock out the RhlC gene, which should yield a strain producing only mono-rhamnolipid. The complementary knockout of RhlB would not be effective because the mono-rhamnolipid is the precursor for the

Review
di-rhamnolipid. The gene knockout has been achieved, but thus far, there is no detailed analysis of the effect on production and yield. A strain producing only mono-rhamnolipid would, in combination with a normal strain, allow considerable manipulation of the ratios of the two forms of biosurfactant. Genetic manipulation techniques are currently being applied to sophorolipid production by the yeast C. bombicola in an effort to produce surfactants tailored to meet specic needs [45,46]. By combining different approaches, it is thus possible to modify both the hydrophilic and hydrophobic portions of the molecule. Although the best studied producers of MELs are the basidiomycetous yeasts of the genus Pseudozyma, Ustilago maydis is also an effective producer under conditions of nitrogen limitation [47]. The gene cluster coding for MEL biosynthesis in U. maydis comprises the mat1 acetyltransferase gene, the mmf1 gene, which species a member of the major facilitator family, mac1 and mac2, encoding putative acyltransferases, and the glycosyltransferase gene emt1. Deletion of the mat1 gene yields nonacylated MELs using this strategy [48], which offers another alternative means of producing a modied product for potential applications which for example require greater water solubility. Production and cost issues Whatever the perceived efcacy of biosurfactants in small scale experiments and trials, their adoption as components of large-volume commercial products will be eventually dictated by cost and production issues (Box 3). The rst issue to consider is the one of safety. So far, there has been no suggestion that any of the biosurfactants investigated, and certainly not the main ones currently under investigation, that is, sophorolipids, rhamnolipids, and MELs, have any major safety or health issues. There have been reports of rhamnolipids acting as immune modulators (e.g., [49]), and they have also been shown to act as virulence factors in P. aeruginosa infections (e.g., [50]). The only reservation lies with rhamnolipids and the main organism that produces them, P. aeruginosa, which in the UK is classied as a class II pathogen. Class II pathogens are not highly infective and can be considered opportunistic pathogens, however, large-scale fermentation production would require some special measures to be taken and care taken with employees involved in the production. Having said that, commercial-scale production is already being undertaken in the USA; particularly for rhamnolipids, and at a company producing food additives (Jeneil Biotech, Milwaukee, USA; www.jenielbiotech.com), with no reported problems. The other biosurfactants, which are produced by yeasts, do not have pathogen issues and commercial-scale production of sophorolipids is also already underway in Asia. Other major production concerns relate to the yields of biosurfactants produced, the substrates needed to produce them [48,51], and the downstream processing required. At present, sophorolipids and MELs can be produced with yields >100 g/l [52], whereas laboratory strains of P. aeruginosa produce only 1020 g/l of rhamnolipids. Information about whether the strains used to produce

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Box 3. Manufacture of biosurfactants

Several companies in different countries are now manufacturing biosurfactants on various scales. Rhamnolipids are produced by at least two companies in the USA using strains of P. aeruginosa. AGAE Technologies (www.agaetech.com) is producing small quantities of highly purified rhamnolipids using strain NY3, and although full details of the process are not declared on their website, it appears that glycerol is the probable major carbon substrate, and yields of about 12 g/l are achieved. The final product is stated to be 95% pure. Larger production is being carried out by Jeneil Biotech (www.jenielbiotech.com) which is a general food additive company. The rhamnolipid products offered by Jeneil range from the crudest preparation comprising fermentation broth with approximately 2% rhamnolipids to partially purified products with up to 99% rhamnolipids. From this information, we can deduce that the yields are again in the 1020 g/l range and that the organism being used may not be a hyperproducer. Sophorolipids are already produced by several companies in, for example, France, Japan, and Korea, with the material being used in products such as dishwasher formulations and Yashinomi vegetable wash. Saraya Co. Ltd. (worldwide.saraya.com) in Japan manufactures sophorolipids using Pseudozyma with palm oil as the main fermentation substrate. Yields for the sophorolipids are not declared but can be expected to be in the 30100 g/l range. Ecover (www. Ecover.com) also markets some products that contain Candida Bombicola/Glucose/Methyl Rapeseedate Ferment, that is, sophorolipids, whereas MG Intobio (http://mgintobio.en.makepolo.com) in Korea markets soaps containing sophorolipids specifically for acne treatment. The French company Soliance (www. soliance.com) also produces sophorolipids from a rapeseed fermentation for cosmetic applications in skin care through antibacterial and sebo regulator activity.

rhamnolipids commercially perform signicantly better than this is not generally available, although there have been some reports of over-producer strains [53]. One big advantage of the glycolipid biosurfactants is that they can be produced from a range of renewable substrates; some of which could be considered waste materials. The separation and purication of low molecular weight glycolipids is relatively straightforward [3], although the process is made more complicated if an oily substrate is used and if quantities of the substrate remain unused after the fermentation. The application of economic technologies based on utilisation of waste substrates for biosurfactant production and the utilisation of cheaper renewable substrates may signicantly contribute to cost reduction [48]. One attractive option as a substrate is glycerol, which is now available in large quantities as a byproduct of the esterication step in biodiesel production from plant glycerides. Eventually, however, biosurfactants will need to be produced in sufcient quantity and at an attractive price to compete with chemical surfactants like LAS, before they will become a major replacement for the surfactants currently used. Concluding remarks Biosurfactants appear to have reached a critical stage in their commercial exploitation; after many years in which interest in them was at a low level, they have now come to the top of the agenda of many companies as a result of the sustainability initiative and green agendas. Potential areas for use are expanding rapidly and useful outcomes will depend on whether biosurfactants can be tailored for specic applications, and whether they can be produced at
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a price that will make them attractive alternatives to chemical surfactants. Several issues do, however, need to be dealt with before large-scale exploitation can take place. In the case of rhamnolipids, the two problems that need to be overcome relate to safety and yield. Despite the published effects of rhamnolipids on the immune system and their role as virulence factors, there are unlikely to be any issues with using these biosurfactants in several products, particularly cleaning and laundering products. The problem of the pathogenic status of the producer organism, P. aeruginosa, is less easily dealt with, although clearly some companies have overcome the problem and the identication of potential new nonpathogenic producer organisms offers a potential solution, providing the products are suitable and the yields are acceptable. The rhamnolipid production in P. aeruginosa is under tight control by the quorum sensing mechanism and this has so far prevented hyperproducing strains being developed, either by mutagenesis and selection or by genetic manipulation. Failure to achieve high yields may eventually preclude rhamnolipids from use in many possible applications. Sophorolipids and MELs by contrast appear to have much greater potential because they have no obvious safety issues, can be produced in high yield. The fact that they have already been included in several commercial products testies to their potential for further exploitation. Thus, there do not seem to be any major impediments to the use of biosurfactants in a wide range of products and applications within the next few years, and we may expect to see an increasing range of domestic products containing at least sophorolipids and MELs on supermarket shelves.
References
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