Microbial Biosurfactants Challenges

8/13/2019 Microbial Biosurfactants Challenges

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Microbial biosurfactants: challengesand opportunities for future

exploitationRoger Marchant and Ibrahim M. Banat

School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, UK

The drive for industrial sustainability has pushed biosur-

factants 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 feed-

stocks. Biosurfactants are also attractive because they

are less damaging to the environment yet are robustenough for industrial use. The most promising biosur-

factants at the present time are the glycolipids, sophor-

olipids produced by Candida yeasts, mannosylerythritol

lipids (MELs) produced by Pseudozyma yeasts, and

rhamnolipids produced by Pseudomonas. Despite the

current enthusiasm for these compounds several resid-

ual problems remain. This review highlights remaining

problems and indicates the prospects for imminent com-

mercial exploitation of a new generation of microbial

biosurfactants.

The move towards biosurfactants

Chemical

surfactants

have

a

major

impact

on

all

our

livesbecause they comprise a major component of many of the

everyday products we use. These chemical surfactants,

many ofwhich are alkyl sulfates or sulfonateswith 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 addi-

tives, cosmetics, and pharmaceuticals, used in agro food

processing and used in the petroleum industry. The world-

wide use of surfactants has grown enormously over the

past few decades, although exact figures for production are

difficult 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 mole-

cules produced principally by microorganisms from sus-

tainable feedstocks. These molecules have the added

advantage that, although they are stable at relatively high

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 be-

fore more widespread use can be envisaged. These pro-

blems relate to yield and cost of production, includingdownstream processing, but also to the tailoring of the

molecules to specific 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, emulsification, wetting, and foaming and depend

on the exact structure of the individual molecules (Box 1).

Microbially produced biosurfactants can be broadly classi-

fied into low molecular weight (glycolipids, lipopeptides,

and flavolipids) [3] and high molecular weight molecules

(polysaccharides,

proteins,

lipopolysaccharides,

and

lipo-proteins) [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 produc-

tion 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

biosur-factants is in the area of laundry products. At present, the

surfactant content of the liquids and powders manufac-

tured is largely alkyl sulfonates such as linear alkylben-

zene 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

Review

Corresponding author: Marchant, R. ([email protected]).

Keywords: rhamnolipids; sophorolipids; mannosylerythritol lipids; MEOR; biofilms.

558 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
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of sophorolipids, although the alkyl chain length is consis-

tent, the degree of unsaturation is not and the number of

acyl groups varies from none to two, with two major con-

figurations 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 surfac-

tants [6,7]. Although the neutron beam scattering tech-

nique 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]. Interest-

ingly, 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 al-

ready been incorporated in some domestic products pro-

duced in Korea.

Another major candidate to be considered for use in this

field is the rhamnolipids produced by P. aeruginosa. Once

again, several different molecules are produced by this

bacterium, withdiffering alkyl chain lengths ranging from

8 to 12 carbon atoms, although two major molecules are

produced, the mono-rhamnolipidwithtwo 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, al-

though thismethodology has been used to investigate the

behaviour of rhamnolipids using the neutron beam scat-

tering

technique

in combinationwith 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 com-

mercial applications.This aspectwillbe dealtwith further

under the section on designer biosurfactants. One signif-

icant problem with the rhamnolipids until recently was

the fact that they were only known to be produced byP.

aeruginosa, a class II opportunistic pathogen; something

that provides a disincentive for large-scale production.

Recently, two nonpathogenic, related bacteria have

been identified as rhamnolipid producers, although the

rhamnolipids produced are different to those produced

byP. aeruginosa.Pseudomonas chlororaphis produces only

mono-rhamnolipid [11], whereasBurkholderia thailanden-

sis 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 specific rhamno-

lipids 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 sen-

sitivity has become a low priority with the drive to reduce

washing temperatures as an energy saving measure.

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, emulsi-

fiers, 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 tobe 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

architectureof the surfactant, these micelles maybe spherical, worm-

like, 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]. Thecritical micelle concentration (CMC)is definedas

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 72mN/m for purewater to a

minimum possible value of approximately

29 mN/m [1].

Once theCMC is reached, ST remains more or less constant.ST and interfacial

tension (IT) between liquid phases are usefulmeasures to determine

whether a microbial culture is producing biosurfactant, but cannot be

used in a quantitativemanner, because once theminimum STorIT 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]. Itis this fact that is driving the search for designer biosurfactants

and forways of producingsingle biosurfactant moleculesrather than

mixtures. Thebehaviour of biosurfactants in solution andat surfaces

can be investigated using techniques such as small-angle neutron

scattering (SANS) [6,7,9,10], although this requiresmajor equipment

facilities.

Congener structure

1 Acidic, C18:1 6.52 Acidic, C18:1, 1Ac 4.93 Acidic, C18:2, 2Ac 2.84 Acidic, C18:1, 2Ac 48.15 Acidic, C18:0, 2Ac 2.86 Lactonic, C18:1, 1Ac 3.06

7 Lactonic, C18:2, 2Ac 2.7




11 Lactonic, C18:1, 2Ac 10.0


HO

HO

COOH

(CH2)n

(CH2)n

O O CH

CH3

OH

CH2OR 1

O

O

CH2OR 2

OH

OH

O

CH2OR 2

OH

OH

O

O O CH

CH3

OH

CH2OR 1

O

C O

HO

(a)(b)

(c)

% Abundance

TRENDS in Biotechnology

Figure 1. (a) Representative chemical composition of sophorolipid mixture

produced by Candida apicolaATCC 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 self-

assembly in solution and also interact differently at surfaces.

Review Trends in Biotechnology November 2012, Vol. 30, No. 11

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The final group of microbial glycolipids with perceived

potential

in this area are the MELs

produced

by

the basid-iomycetous yeasts of thegenusPseudozyma and also by the

fungus Ustilago. MELs fromPseudozyma have been exten-

sively investigated [13]. As with the other producer organ-

isms, a range of different MEL molecules are produced,

differing in alkyl chain length and degree of acylation.

Onemajor advantageof theMELproducers, likethe sophor-

olipid producers, is that resting cells continue to synthesize

the biosurfactant, allowing yields to exceed 100 g/l [14].

Biofilm prevention and disruption

Althoughmuchof the laboratory-basedworkwith bacteria is

conducted with planktonic cultures, mixed species biofilms

are a more common mode of growth for these organisms.

Biofilms havea complex structure that allows cell communi-

cation, (quorum sensing), to take place, which also acts as a

protection for the cells from external factors such as anti-

biotics [15]. Biofilms can develop on a wide range of surfaces

includingdomestichouseholdareasandmedicaldevicessuch

as catheters and prostheses. Biosurfactants are believed to

play a major role in the development and maintenance of

biofilms in P. aeruginosa [16]; partly at least through the

maintenance of water channels through the biofilm. Atten-

tion is now turning to the possibility that biosurfactants can

be used to disrupt established biofilms and to prevent the

development of new ones. Rhamnolipids can inhibit the

adhesion of yeasts and bacteria to voice prostheses [17]

and can mediate the disruption of established biofilms

[1821].

The

lipopeptide surfactants

putisolvin

I

and

II

pro-duced byPseudomonasputida are able to inhibit the forma-

tion of biofilms of other Pseudomonas strains and indeed to

break down established biofilms [22]. Although it is usefulat

a preliminary stage to examine the effect of the biosurfac-

tants alone on biofilms, the next step must be to determine

the interactions between biosurfactants and other compo-

nents of cleaning agents suchas chemical surfactants.More-

over,pHand othercompoundsmightboostactivity,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 zoos-

pores used as a bioassay. The interesting question, howev-

er, is whether more resistant cells, for example, bacteria

with cell walls, may be killed by biosurfactants. For exam-

ple, sophorolipids improve sepsis survival in model sys-

tems 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 encourag-

ing results have been reported using low concentrations

(0.1%) to treat ulcers and burns. This area of study cer-

tainly warrants further investigation and extension to

ST5HEXEXTRACT #16 RT:0.010.09 AV:6 NL:4.64E5F:c ms[175.001000.00]

100

95

90

85

80

75

70

65

60

55

50

Relave

abundance

45

40

35

30

25

20

15

10

5

0

200 250 300 350 400 450 500 550 600m/z

650 700 750 800 850 900 950 1000

989.2

975.1

955.2915.0845.0815.7

762.6

761.5

747.4678.1

711.8

677.3

622.1

588.4561.2

531.2

504.3

503.2

649.2

Rha-C10-C10

Rha-Rha-C10-C10

475.1

457.0437.5359.0

333.1

325.3

303.1289.9248.6195.9

650.3

OHHO

O

OCH3

CH3

HO

HO

O

O OO

OH

O

HO

CH3

CH3

OHOH

Monorhamnolipid

Dirhamnolipid

HOH3C

O

O

O C OO C OH

H3C

H3C


Figure 2. Mass spectroscopy data showing the range of rhamnolipid congeners produced by Pseudomonas aeruginosastrain ST5 with the two main products the mono

and di-rhamno forms with two C10 alkyl chains.


560


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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 bio-

surfactants produced by microorganisms; one of which is

their

involvement

in

the

metabolism

of

hydrophobic

sub-strates [29]. In aqueous environments, the interfacial ac-

tivity of biosurfactants and bioemulsifiers can make

substrates like hydrocarbons more amenable to the degra-

dative activity of the cell. This being the case, we might

expect that the majority of bacteria that utilise hydropho-

bic 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 hydro-

phobic substrate. The obvious situation where this might

be advantageous would be in the field of bioremediation,

particularly in situ bioremediation. The mechanisms in-

volved in interactions between biosurfactants or the mi-

crobial cells and immiscible hydrocarbons include: (i)emulsification; (ii) adhesion/de-adhesion of microorgan-

isms to and from hydrocarbons; (iii) micellarisation; and

(iv) desorption of contaminants; all ofwhich are expected to

enhance the rates of biodegradative bioremediation. Cur-

rent literature generally supports such conclusions, how-

ever, some cases in which complex interactions among

microbial cells, organic substrates, surface active com-

pounds 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 contamina-

tion are the thermophilic bacilli of the genus Geobacillus

[31], which

do

not

produce

any

biosurfactant.

These

organ-isms 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 environ-

ment allows them to become active and to compete effec-

tively 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 degra-

dation rate and extent of selected hydrocarbons was

achieved when both the nutrient supplements and biosur-

factant were added [35,36]. It is therefore clear that the

addition of biosurfactants, even to organisms that do not

produce their own, canhavehighly beneficial effects.At the

present time, marine and coastal oil spills are treated, at

least in part, by the use of chemical surfactants and

emulsifiers, future investigation of the use of biosurfac-

tants in their place is certainly a fruitful avenue for inves-

tigation.

Biosurfactants also have extensive potential application

in the petroleum industry, which in turn affects the envi-

ronment (Box 2). Microbially enhanced oil recovery

(MEOR) is a technique that eitheruses 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.

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 techni-

ques

[37]. A

form

of

MEOR

has

been

pioneered

effectivelyat 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 in-

volving the use of chemical or physical processes such as

pressurisation, water flooding, or steaming, are often in-

applicable 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 difficult to dispose ofwithout

adversely affecting the environment [40]. This is particu-larly the case in marine environments where the use of

biodegradable biosurfactants rather than chemical surfac-

tants would have major environmental benefits.

Designer biosurfactants

Microbial biosurfactant producers invariably give a prod-

uct 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 specific properties for the surfactant used. The

ability

to

select

or

design

specific

biosurfactants

is

there-fore highly desirable. As we can see, isolation and purifi-

cation of individual components is feasible but unlikely to

be economic on a large scale [3]. The next simplest ap-

proach is to modify growth and production conditions or to

select specific strains of the producer organisms. In prac-

tice 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 sophor-

olipids [41].

One simple and effective approach to biosurfactant

modification used a naturally produced acylated MEL

and removed the acyl groups with a lipase-catalysed hy-

drolysis, producing a nonacylated product (MEL-D) [42].

They are able to show a higher critical aggregation con-

centration and excellent surface tension, lowering capacity

for the deacylated MELs, indicating that the new MEL-D

mayhaveapplications infields inwhich a lamellar-forming

glycolipid is required.

A more difficult and costly strategy is to investigate

genetic modification of the producer organisms. The syn-

thetic 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


561


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to contemplate cloning the pathway into another host

bacterium, for example, Escherichia coli. RhlA and RhlBgenes 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 di-

rhamnolipid. This was indeed the outcome but with only

small yields recorded. It does seem unlikely that this

approach can be completely successful because production

of large quantities of biosurfactant depends on the meta-

bolic fluxwithin 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 com-

plementary knockout of RhlB would not be effective

because the mono-rhamnolipid is the precursor for the

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 characterist ics 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 CO2injection, alkaline surfactant

polymers (ASPs), solvents or steam to drive out an additional 1520%

through sweeping the oil towards the producingwells 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.

Severalmethods, includingMEOR,havebeengaining 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

throughpartial breakdown of thelargemolecular structureof crudeoil,

making itmore fluid; production ofCO2 gasas a byproductof microbial

metabolism,which both pressurises the reservoir andmoves upward,

displacing oil in the well; production of biomass that accumulates

between the oil and the rock surface of the well, physically displacing

theoil andmaking it easier to recover from thewell; 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 sothat 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 andhave several environmental advantages; theyare achieved

either through ex situproduction and injection into oil reservoirs, or

through injection of selected microorganisms to produce biosurfac-

tants 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

(Bacteria, nutrients,

and/or biosurfactants)

Pressing watercontaining microorganisms

biosurfactants nutrients

Enhancedmobility

Biodegradaonof crude oil (to lowmolecular weight)

Microbial metabolites/biosurfactants

Crude oil Advanced waterImprovement of crude oil mobility

Improvement of oil reservoirpercolaon

Enhanced oil recovery

GasAcid

BiomassPolymer

Producon well


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


562


6/8

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-rham-

nolipid 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.

bombi-cola in an effort to produce surfactants tailored to meet

specific 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 genusPseudozyma, Ustilago

maydis is also an effective producer under conditions of

nitrogen limitation [47]. The gene cluster coding for MEL

biosynthesis inU. maydis comprises the mat1 acetyltrans-

ferase gene, the mmf1 gene, which specifies 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 alter-native means of producing a modified product for potential

applications which for example require greater water sol-

ubility.

Production and cost issues

Whatever the perceived efficacy 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 first

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 investi-

gation,

that

is,

sophorolipids,

rhamnolipids,

and

MELs,have any major safety or health issues. There have been

reports of rhamnolipids acting as immunemodulators (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

classified as a class II pathogen. Class II pathogens are not

highly infective and can be considered opportunistic patho-

gens, 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 under-

taken in the USA; particularly for rhamnolipids, and at a

companyproducing food additives (JeneilBiotech,Milwau-

kee, USA; www.jenielbiotech.com), with no reported pro-

blems. 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. aer-

uginosa produce only 1020 g/l of rhamnolipids. Informa-

tion about whether the strains used to produce

rhamnolipids commercially perform significantly 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

separationand purification 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 sub-

strates may significantly 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

esterification step in biodiesel production from plant gly-

cerides. Eventually, however, biosurfactants will need to

beproduced in sufficient quantity andatanattractiveprice

to compete with chemical surfactants like LAS, before they

will become a major replacement for the surfactants cur-

rently 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

specific applications, and whether they can be produced at

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 quan-

titiesof highly purified rhamnolipidsusing strainNY3, andalthough

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 12g/l are achieved. The final product is stated to be95% 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 vegeta-

ble 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 containCandida 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.


563
http://www.jenielbiotech.com/http://www.agaetech.com/http://www.jenielbiotech.com/http://www.%20ecover.com/http://mgintobio.en.makepolo.com/http://mgintobio.en.makepolo.com/http://www.%20soliance.com/http://www.%20soliance.com/http://www.%20soliance.com/http://www.%20soliance.com/http://mgintobio.en.makepolo.com/http://mgintobio.en.makepolo.com/http://www.%20ecover.com/http://www.jenielbiotech.com/http://www.agaetech.com/http://www.jenielbiotech.com/


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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 asvirulence factors, there are unlikely to be

any

issues

with

using

these

biosurfactants

in

several

pro-ducts, 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 iden-

tification of potential new nonpathogenic producer organ-

isms 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 muta-

genesis and selection or by genetic manipulation. Failure

to achieve high yields may eventually preclude rhamnoli-

pids from use in many possible applications. Sophorolipids

and MELs by contrast appear to have much greater poten-tial 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 testifies to

their potential for further exploitation. Thus, there do not

seem to be any major impediments to the use of biosurfac-

tants 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 sophoroli-

pids and MELs on supermarket shelves.

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