Biotechnology and Molecular Biology Reviews Vol. 3 (5), pp.

111-117, October, 2009

Available online at http://www.academicjournals.org/bmbr
ISSN 1538-2273 © 2009 Academic Journals

Standard Review

An investigation for potential development on

biosurfactants
A. Salihu1*, I. Abdulkadir2 and M. N. Almustapha3
1*
Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria.
2
Department of Chemistry, Ahmadu Bello University, Zaria, Nigeria.
3
Department of Pure and Applied Chemistry, Usman Danfodio University, Sokoto, Nigeria.
Accepted 3 September, 2009

Biosurfactants are surface-active metabolites produced by microorganisms. The applications of these

biological compounds in the field of enhanced oil recovery and bioremediation proved effective.
Besides their environmental applications; biosurfactants have shown interesting properties in several
processes. Thus, this article attempts to organize this Information for ease of reference and further
stimulates those that have interests in the area to explore further especially in biodegradation of
recalcitrant compounds.

Key words: Biosurfactant, bioremediation, biodegradation.

INTRODUCTION

Biosurfactants have received more and more attention in tive in stabilizing oil-in-water emulsions (Neu, 1996;
recent years as surface-active compounds released by Franzetti et al., 2009). These high molecular weight
microorganisms that have some influence on interfaces, biosurfactants are highly efficient emulsifiers that work at
most notably on the surface tension of liquid - vapor low concentrations and exhibit considerable substrate
interfaces. They are interesting amphiphilic molecules of specificity (Dastgheib et al., 2008).
microbial origin; whose hydrophobic and hydrophilic do- However, general classification based on parent chemi-
mains depend on the carbon substrate and the organism cal structure and surface properties are represented in
strain. They have various biological functions/ properties the following groups:
and have potentials in commercial applications in the
food, microbiological, pharmaceutical and therapeutical 1. The glycolipids, in which carbohydrates such as
agents in biological industries, as a bio-control agent in sophorose, trehalose or rhamnose are incorporated to a
agricultural applications and in health and beauty long-chain aliphatic acid or lipopeptide. This is seen in
products for the cosmetic industries (Nayak et al., 2009; rhamnolipids produced by Pseudomonas aeruginosa
Mukherjee et al., 2009; Tugrul and Cansunar, 2005; which constitute one or two sugar moieties linked to
Benincasa et al., 2004; Volkering et al., 1998; Fiechter, caprilic acid group by a glycosidic bond (Rosenberg and
1992). Many microorganisms have ability to produce a Ron, 1999).
wide range of biosurfactants, as such initial classification 2. Biosurfactant with amino-acid moieties such as sur-
was made into two; based on molecular weights, factin produced by Bacillus subtilis composed of several
properties and cellular localizations. The low molecular amino-acid structures linked to a molecule of 3-hydroxy-
weight biosurfactants e.g. glycolipids, lipope-ptides, 13-methyl tetradecanoic acid.
flavolipids, corynomycolic acids and phospholipids lower 3. The emulsan synthesized by Acinetobacter calcoa-
the surface and interfacial tensions at the air/water ceticus RAG-1 is an extracellular polysaccharide - lipid
interfaces while the high molecular weights are called complex (Rosenberg et al., 1979).
bioemulsans, (such as emulsan, alasan, liposan, poly- 4. Protein-like substances (e.g. liposan) constitute small
saccharides and protein complexes) and are more effec- amount of carbohydrates as produced by Candida
lipoltica (Calvo et al., 2008).

Biosurfactants have unique advantages which include:

*Corresponding author. E -mail: [email protected]. structural diversity that may lead to unique properties
112 Biotechnol. Mol. Biol. Rev.

Figure 1. Chemical structures of some common biosurfactants (a) Mannosylerythritol lipid (b) Surfactin (c) trehalose lipid (d)
Sophorolipid (e) Rhamnolipid (f) Emulsan.

(chemical structures of commonly studied biosurfactant substrates, such as molasses (Makkar and Cameotra,
are shown in Figure 1); the possibility of cost effective 1997) agro-industrial wastes,( Makkar and Cameotra,
production and their biodegradability. These properties 1999), soapstock and a by-product of the vegetable oil
make them a promising choice for applications in enhan- refining processes (Benincasa et al., 2004), are surfactin
cing hydrocarbon bioremediation (Whang et al., 2008). produced by B. subtilis and rhamnolipids produced by P.
Biosurfactants are able to retain their properties even aeruginosa. Others produced by different species of
under extreme conditions of pH, temperature, salinity microorganisms are presented in Table 1. The foci for
(Ron and Rosenberg, 2002) and have low irritancy and reduction of biosurfactant production costs are the
compatibility with human skin (Pornsunthorntawee et al., microbes (selected, adapted, or engineered for high
2009). yields of product), the process (selected, designed and
Other important advantages, such as bioavailability, engineered for low capital and operating costs), the
activity under a variety of conditions, ecological accepta- microbial growth substrate and/or the process by-
bility, low toxicity, their capacity to be modified by bio- products (minimized or managed as saleable products
technology and genetic engineering and their capability of rather than treated and discarded as wastes).
increasing the bioavailability of poorly soluble organic
compounds, such as polycyclic aromatics are among the
unique properties of these agents (Tugrul and Cansunar, Synthetic surfactants
2005). Thus, their use could offer some solutions in
bioremediation of contaminated soil and subsurface Conventional chemical surfactants have been extensively
environments (Lai et al., 2009). Also, biosurfactants could used and their derivatives are costly and of serious
easily be produced from renewable resources via environmental concern; they are potential threats to the
microbial fermentation, making them have an additional environment due to their recalcitrant and persistent
advantage over chemically synthetic surfactants. The nature. Therefore, with current advances in biotechno-
important challenges for the competitive production of logy, attentions have been paid to the alternative
biosurfactants include high yields, alternative low-cost environmental friendly processes for production of
substrates and cost-effective bioprocesses different types of biosurfactants from microorganisms
(Pornsunthorntawee et al., 2009). Some of the biosur- (Lotfabad et al., 2009).
factants that have been studied using alternative low-cost Also, as part of the problems; most manufacturers of
 Salihu et al. 113

Table 1. Some of the common biosurfactants with their producing microorganisms.

Organisms Biosurfactants Sources References

Pseudomonas aeruginosa Rhamnolipids Petrochemical Whang, et al., 2008; Haba,
wastewater, soap stock, et al., 2003; Wei, et al.,
Petroleum contaminated 2008.
soil, etc.
Pseudoxanthomonas spp Nayak, et al., 2009.
PNK-04

Pseudomonas alcaligenes Oliveira, et al., 2009.

Rhodococcus erythropolis Trehalose lipids Oil contaminated soil, Marquez, et al., 2009;
51T7 Micrococcus luteus Hydrocarbon gas station Tuleva, et al., 2009.
BN56 soil, etc.

Bacillus subtilis ATCC Surfactin Petroleum sludge, Pornsunthorntawee, et al.,

21332 Waste soybean oil, etc. 2008; Lee, et al., 2008.

Candida antarctica Mannosylerythritol lipids Vegetable oil and Kim, et al., 2002;
soybean oil waste Kitamoto, et al., 2001.

Gordonia spp BS29 Bioemulsan Diesel contaminated soil Franzetti, et al., 2009.

chemical surfactants set the recommended dispersal the nitrogen source as well as the C: N ratio, nutritional
ratios for their products on the basis of the economics limitations and chemical and physical parameters such as
and effectiveness of the dispersant with minimal conside- temperature, aeration, divalent cations and pH influence
ration for the potential harm that can be caused in the not only the amount but also the type of polymer
receiving ecosystem (Laux et al., 2000; Putheti and Patil, produced (Toledo et al., 2008; Lotfabad et al., 2009). The
2009). However, concern still exists on the possible toxic commonly used carbon sources include carbohydrates,
effects of these surfactants on aquatic organisms, espe- hydrocarbons and vegetable oils. It has been concluded
cially if they are used in near shore waters (Otitoloju and from a number of studies that different carbon sources
Popoola, 2009; Venosa and Holder, 2007). can influence the composition of biosurfactant formation.
The effects of chemical surfactants on biostimulation of Arthrobacter produces 75% extracellular biosurfactant
indigenous microorganisms in enhancing the removal of when grown on acetate or ethanol but it is totally
organic pollutants yielded inconsistent results. Decrease extracellular when grown on hydrocarbon (Mulligan and
in the rate of biodegradation of organic pollutants espe- Gibbs, 1993; Putheti and Patil, 2009). The nitrogen
cially at higher concentrations as suggested by Sun et al. source in the medium plays a significant role in produc-
(2008) may be linked to the interaction of surfactant with tion and contributes to pH control. Several organic and
the lipid membrane and their effects on enzymes and inorganic sources proved effective. Whang et al. (2009)
other cellular proteins necessary for basic functions of the showed that ammonium concentration and pH enhanced
microorganisms. Thus, the use of biosurfactants in place biosurfactant efficiency and increase emulsification ability
of chemical surfactants can minimize all sets of threats on diesel oil, thus establishing the optimum pH for the
posed by the latter. production to be towards neutrality. Environmental factors
Despite all these effects; chemical surfactants find such as temperature, pH, agitation and oxygen availabi-
applications in different operations, such as drilling, lity also affect the production of biosurfactant (Banat,
cement production, slurries, fracturing, acidization, 1995).
demulsification, corrosion inhibition, transportation, clean-
ing, water flooding, chemical, foam and steam flooding
and environment protection as oil spill dispersants (Atta METHODS USED FOR IDENTIFICATION
et al., 2006).
Several methods for screening and estimation of
biosurfactants have been developed.
Factors affecting biosurfactant production Drop-collapse method is one of the qualitative methods
used to determine the presence of biosurfactant. Tugrul
Composition and emulsifying activity of the biosurfactant and Cansunar (2005) conducted experiments to confirm
depend not only on the producer strain but also on the the reliability of the method using polystyrene microwell
culture conditions. Thus, the nature of the carbon source, plate; oil-coated wells collapse was observed when the
114 Biotechnol. Mol. Biol. Rev

2008).
In contrast to the methods described so far, high perfor-
mance liquid chromatography (HPLC) is not only
appropriate for the complete separation of different
biosurfactants, but can also be coupled with various
detection devices (UV, MS, evaporative light scattering
detection, ELSD) for identification and quantification of
biosurfactants (Heyd et al., 2008).
Das and Mukherjee (2005, 2007) described the acidifi-
cation of the solution containing biosurfactant in the
presence of HCl and equal volume of ethyl acetate in a
separatory funnel. Organic layer is collected following
phase separation and further dehydrated in rotatory eva-
porator. The crude sample is then dissolved in sodium
Bicarbonate and filtered; adjusting the pH with HCl;
precipitation occurs overnight at 4°C. The pure product of
biosurfactant is quantitatively obtained by centrifugation
Figure 2. Relationship of surface tension, interfacial tension and
and freeze-drying.
the CMC with surfactant concentration (Mulligan, 2005).

Biosurfactants enhanced biodegradation of

culture broth contained biosurfactant and there was no hydrocarbon compounds
change in the shape of the droplets in the absence of
biosurfactant. Itoh and Suzuki (1972) were the first to show that
Thin layer chromatography (TLC) is also used in hydrocarbon culture media stimulated the growth of a
preliminary characterization of the biosurfactant where rhamnolipid producing strain of P. aeruginosa. Recent
the cell free extract containing biosurfactant is separated researches confirmed the biosurfactant effects on
on a silica gel plate using chloroform: methanol: water hydrocarbon biodegradation by increasing microbial
(70:10:0.5, v/v/v); this is then followed by using colour accessibility to insoluble substrates and thus enhance
developing reagents. Lipopeptide biosurfactant showed their biodegradation (Zhang and Miller, 1992; Hunt et al.,
red spots in the presence of ninhydrin reagent, while 1994).
glycolipid biosurfactant is detected as yellow spots when Various experiments have been conducted that showed
anthrone is used as the colour reagent (Yin et al., 2009). the effects of biosurfactants on hydrocarbons; enhancing
Additionally, blood agar hemolysis tests is another their water solubility and increasing the displacement of
method used; where the organisms with biosurfactant oily substances from soil particles. Thus, biosurfactants
ability are streaked on blood agar plates and incubated at increase the apparent solubility of these organic
40°C. The plates are visually monitored for the presence compounds at concentrations above the critical micelle
of clearing zone around the colonies which is indicative of concentration (CMC), which enhance their availability for
surfactant biosynthesis. The diameter of the clear zones microbial uptake (Chang et al., 2008). For these reasons,
depends on the concentration of the biosurfactant inclusion of biosurfactants in a bioremediation treatment
produced (Youssef et al., 2004; Ghojavand et al., 2008). of a hydrocarbon polluted environment could be really
Surface tension measurement by a du Nöuy ring-type promising, facilitating their assimilation by microorga-
tensiometer (Krüss, K10T) is one of the simplest techni- nisms (Calvo et al., 2008).
ques used. The surface tension measurement is carried Many of the biosurfactants known today have been
out at room temperature after dipping the platinum ring in studied to examine their possible technical applications
the solution for a while in order to attain equilibrium (Nayak et al., 2009). Most of these applications involve
conditions. A higher biosurfactant concentration in the their efficiency in bioremediation, dispersion of oil spills
test sample provides a lower surface tension until the and enhanced oil recovery. Alcanivorax and Cyclocla-
critical micelle concentration (CMC) is reached. The CMC sticus genera are highly specialized hydrocarbon degra-
is obtained where the surface tension remains steady ders in marine environments. Alcanivorax borkumensis
despite the changes in concentration as represented in utilizes aliphatic hydrocarbons as its main carbon source
Figure 2 (Desai and Banat, 1997; Crosman et al., 2002). for growth and produces an anionic glucose lipid biosur-
This is widely used because of its accuracy, eases to use factant and thus potentials of Alcanivorax strains during
and provides a fairly rapid measurement of surface and bioremediation of hydrocarbon pollution in marine
interfacial tension. In general, biosurfactants produced by habitats have been studied (Olivera et al., 2009); thus,
P. aeruginosa strains have the ability to reduce the this property needs to be studied extensively in soil to
surface tension of water, when measured with a tension- ensure its effectiveness.
metre from 72 to 30 mN/m (Pornsunthorntawee et al., Several species of P. aeruginosa and B. subtilis pro-
 Salihu et al. 115

duce rhamnolipid, a commonly isolated glycolipid biosur- antimicrobial, emulsifier and conservative agent in agro-
factant and surfactin, a lipoprotein type biosur-factant chemical, food industries, as well as in bioremediation
respectively; these two biosurfactants have been shown applications.
by Whang et al. (2008), to increase solubility and These surface-active compounds enhanced the bio-
bioavailability of a petrochemical mixture and also stimu- availability of recalcitrant and hydrophobic organic
late indigenous microorganisms for enhanced biodegra- pollutants through different mechanisms as suggested by
dation of diesel contaminated soil. Gordonia species Volkering et al. (1998):
BS29 grows on aliphatic hydrocarbons as sole carbon
source has been found to produce Bioemulsan, which 1. Decrease in interfacial tension between an aqueous
effectively degrade crude oil, PAHs and other recalcitrant and a non-aqueous phase resulting in formation of
branched hydrocarbons from contaminated soils. The emulsions that lead to improved mass transfer of
rate of biodegradation is dependent on the chemico- pollutants to the aqueous phase.
physical properties of the biosurfactants and not by the 2. Formation of micelles results in solubilization of
effects on microbial metabolism (Franzetti et al., 2008; hydrophobic organic compounds; thus the hydrophobic
Franzetti et al., 2009). compounds remain in the core of the micelles whereas
Corynebacterium alkanolyticum produces a phosphor- the hydrophilic molecules interact with the exterior part
lipid biosurfactant with a relatively low yield; however, the (Volkering et al., 1995); however, opinion is divided whe-
use of self-cycling fermentation processes resulted in ther the solubilized hydrocarbons are directly available to
three fold increase in the biosufactant production. Also, the degrading micro-organisms.
the yield could be further increased to five fold by addition 3. Surfactants’ interaction with hydrocarbons in the soil
high amount of the limiting substrates (Crosman et al., through mobilization lowers the surface tension of the
2002) pore water and hydrocarbons in soil particles (Deitsch
Persistent organic pollutants found in oil containing and Smith, 1995).
wastewater and sediments, such as PAHs (phenanth- Finally, more insight is required for large scale use of
rene, crysene), are also hydrophobic in nature and thus biosurfactants, this involves laboratory and field experi-
water solubility of PAHs normally decrease with the ments, especially in situ studies with proper control
increasing number of rings in molecular structure. This processes.
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