REDACTION : BIOSURFACTANTS

INTRODUCTION :
The overall establishment of biosurfactants is well-known to be impeded by a lack of
availability of economic and versatile products. Currently there is only a very limited offer of
commercially available biosurfactants, e.g., surfactin, sophorolipids and rhamnolipids. A variety
of new biosurfactants respectively producing strains are the key issue in overcoming the
economic obstacles of the production of biosurfactants. Therefore, increased efforts in the
discovery of new biosurfactant producing microbes must be made by applying a broad range of
different screening methods, which is the focus of this chapter.
The principle aim in screening for new biosurfactants is finding new structures with strong
interfacial activity, low critical micelle concentration (cmc), high emulsion capacity, good
solubility and activity in a broad pH-range. Besides these physicochemical properties,
commercial viable biosurfactants have to be economically competitive. Therefore, the second
aim in screening is the discovery of good production strains with high yields.
Biosurfactants may be involved in pathogenesis due to their surface activity; however, for
security and regulatory reasons, production strains should be nonpathogenic. In the above
mentioned example of rhamnolipids this is not the case as Pseudomonas aeruginosa, the most
common producing bacteria, is a pathogen.
A variety of methods for the screening of biosurfactant producing microbes has been
developed and successfully applied. Since the 1970s there have been various trials in this field.
These screenings have mostly been limited to a manageable number of samples. In recent years
automation and miniaturization have led to the development of high throughput methods for
screening of biosurfactant producing strains. A broad application of such methods could
eventually lead to the desired upsurge of new commercially interesting strains.
An efficient screening strategy is the key to success in isolating new and interesting microbes
or their variants, because a large number of strains needs to be characterized. A complete
strategy for screening of new biosurfactants or production strains consists of three steps:
sampling, isolation of strains and investigation of strains. Theses steps will be addressed in the
next paragraphs. Bioinformatical approaches like homology search are not included herein.

Définition :
Microbial surface-active compounds
Microbial surface-active compounds are a group of structurally diverse molecules produced by
different microorganisms and are mainly classified by their chemical structure and their
microbial origin. They are made up of a hydrophilic moiety, comprising an acid, peptide cations,
or anions, mono-, di- or polysaccharides and a hydrophobic moiety of unsaturated or saturated
hydrocarbon chains or fatty acids. These structures confer a wide range of properties, including
the ability to lower surface and interfacial tension of liquids and to form micelles and
microemulsions between two different phases. These compounds can be roughly divided into
two main classes (Neu 1996): low-molecular-weight compounds called biosurfactants, such as
lipopeptides, glycolipids, proteins and high-molecular-weight polymers of polysaccharides,
lipopolysaccharides proteins or lipoproteins that are collectively called bioemulsans (Rosenberg
and Ron 1997) or bioemulsifiers (Smyth et al. 2010b). The former group includes molecules
which can efficiently reduce surface and interfacial tension, while the latter are amphiphilic and
polyphilic polymers which are usually more effective in stabilising emulsions of oil-in-water but
do not lower the surface tension as much (Smyth et al. 2010a). The best-studied microbial
surfactants are glycolipids. Among these, the best-known compounds are rhamnolipids,
trehalolipids, sophorolipids and mannosylerythritol lipids (MELs) (Fig. 1), which contain mono-
or disaccharides, combined with long-chain aliphatic acids or hydroxyaliphatic acids.
Rhamnolipid production by Pseudomonas species has been extensively studied, and potential
applications have been proposed (Maier and Soberón-Chávez 2000). Rhamnolipids from
Pseudomonas aeruginosa are currently commercialised by Jeneil Biosurfactant, USA, mainly as
a fungicide for agricultural purposes or an additive to enhance bioremediation activities.
Trehalolipids are produced by a number of different microorganisms, such as Mycobacterium,
Nocardia and Corynebacterium. However, the most extensively studied compounds in this class
are trehalose dimycolates produced by Rhodococcus erythropolis (Rapp et al. 1979).
Sophorolipids, on the other hand, are produced mainly by yeasts, such as Candida bombicola
(also known as Torulopsis bombicola), Centrolene petrophilum, Candida apicola and
Rhodotorula bogoriensis, while MELs are produced by Pseudozyma yeasts, Pseudozyma aphidis,
Pseudozyma antarctica and Pseudozyma rugulosa (Konishi et al. 2007a, b). Cyclic lipopeptides
are produced by a number of Bacillus species as antibiotic molecules. Among these, the most
important compound is surfactin produced by Bacillus subtilis because of its very high activity
(Desai and Banat 1997; Rosenberg and Ron 1999). A wide variety of microorganisms, including
some Archaea, produce high-molecular-weight polymers, the most extensively investigated
being bioemulsans (Fig. 1) which are synthesised by various species of Acinetobacter. The first
studied compound was RAG-1 emulsan, an amphiphilic polysaccharide produced by
Acinetobacter calcoaceticus RAG-1, which is also the only commercially available bioemulsifier
at present (Suthar et al. 2008).
Biosurfactants are amphiphlic molecule produced by a wide variety of plants, animals and
microorganisms (bacteria, yeast and fungi) and the microbial derived surfactants are either
adhere to cell surface or excreted extra-cellularly in the growth medium; contain both
hydrophobic and hydrophilic moieties that confer the ability to accumulate between fluid phases
thus reducing surface and interfacial tension at the surface and interface respectively.[3,4]
Recently, biosurfactants attracted an attention in the past five decades as an improved alternative
to chemical surfactants due to their low toxicity, higher biodegradability, and effectiveness at
extremes of temperature, pH, and salinity. [3] Due to these traits, biosurfactants find widespread
application in the field of bioremediation of pollutants,[5] oil, food, cosmetic, and
pharmaceutical industry.[1,3] During the past few decades biosurfactant production from various
microorganisms has been studied extensively. To the best of our knowledge, very few attempts
have been made to describe the research and development strategies of making the biosurfactant
production process cheaper and commercially attractive. Main aim of this review article is to
emphasise the exploitation of cheap and easily available agro-industrial waste as substrate for
commercial production of biosurfactants along with waste management and their potential
applications in food and agriculture field.
Biosurfactants are a heterogeneous group of microbial metabolites and, therefore, exhibit vastly
different physical properties. Especially with respect to foaming properties, reduction of surface
tension, and emulsification capacities, this has consequences for handling biosurfactant
fermentations in bioreactors. For example, the biosurfactants rhamnolipid, surfactin, and
mannosylerythritol lipids can be grouped into high-foaming biosurfactants, whereas
sophorolipids are low-foaming biosurfactants (Rau et al. 2005; Hirata et al. 2009; Müller et al.
2010). Owing to this diversity, several bioreactors have been reported for biosurfactant
production reaching from “standard” constructions like stirred tank bioreactors (Müller et al.
2010) to “exotic” constructions like the rotating disc bioreactor (Chtioui et al. 2012). This
chapter presents different approaches to encounter the obstacles regarding biosurfactant
production from the perspective of equipment used for cultivation without providing an
economic evaluation.
Origine :
MICROORGANISMES PRODUCTEURS
Bien que diverses espèces de bactéries, de levures et de champignons filamenteux soient
considérées comme des organismes producteurs dans plusieurs brevets liés aux biosurfactants et
aux bioémulsifiants, les espèces appartenant aux Acinetobacter, Bacillus, Pseudomonas,
Torulopsis et Candida viennent au premier plan (Figure 11.4). De plus, dans de nombreux
brevets, en particulier lorsque l'invention est centrée sur la formulation pour certaines
applications et/ou procédés de production, appareil ou processus en aval, le microorganisme
producteur n'est pas spécifié. Dans certains cas, un consortium bactérien d'origine indéterminée a
été utilisé. Les espèces Bacillus versus Pseudomonas sont les producteurs les plus courants dans
les publications de brevets (tableaux 11.1 et 11.2). Les membres du genre Bacillus en forme de
bâtonnet, des organismes chimio-organotrophes, aérobies ou anaérobies facultatifs formant des
endospores résistantes sont utilisés depuis de nombreuses années (Slepecky et Hemphill, 2006).
La surfactine découverte par Arima et al. (1968) à partir du bouillon de culture de Bacillus
subtilis est le biosurfactant le plus actif (un lipopeptide cyclique) produit par le Bacillus
Biosurfactants are natural products derived from bacteria, yeasts or fungi. The complex chemical
structures and physical properties of biosurfactants generally result in properties equal to or
exceeding many synthetic surfactants. Biosurfactants demonstrate low toxicity to freshwater,
marine and terrestrial ecosystems and are potential candidates for a variety of environmental
applications. Research has largely been focused on the enhancement of oil biodegradation and
microbial-enhanced oil recovery. The solubilisation and emulsification of toxic heavy metals by
biosurfactants have also been reported, assisting in the recovery of such hazardous materials
from contaminated sites. The future success of biosurfactant technology in bioremediation
initiatives is promising, but will require the precise targeting of the biosurfactant systems to
reduce production costs and increase product yield. The suitability to the physical conditions and
chemical nature of the pollution-affected site is also important the commercialisation of these
biomolecules.
NATURE :
CLASSIFICATION :
All biosurfactants comprise at least one hydrophilic and one hydrophobic part due to their
amphiphilic character. The molecular structure often also contains several hydrophobic and
corresponding hydrophilic parts. The hydrophobic part usually comprises saturated or
unsaturated fatty acids, hydroxyl fatty acids, or fat alcohols, with various other structures such as
isoprenoids being possible as well. The chain length usually comprises between 8 and 18 carbon
atoms. The hydrophilic part may be made up of either structurally relatively simple ester,
hydroxyl, phosphate, or carboxyl groups, or of carbohydrates—such as mono, oligo, or
polysaccharides—peptides or proteins. Many anionic and neutral biosurfactants are known.
Cationic biosurfactants, in contrast, have been described extremely rarely, probably because they
have a toxic effect, just like cationic surfactants in general. Within the biosurfactants, the
glycolipids form the greatest share, with the non-sugar component, the aglycone, being highly
versatile. These structures are particularly interesting since many biosurfactants exhibit high
efficiency at concurrently good biological degradability. They can also be produced from
renewable resources. Generally, biosurfactants are assigned the following properties beneficial
for industrial use:
• Great structure diversity (about 2000 described biosurfactants) • Beneficial surfactant
properties • Low eco-toxicity • Antibiotic or bioactive effects • Complete biological
degradability • Production from renewable resources
Although the biotechnological production of microbial surfactants has already been established
so far, they have only been used in niche areas due to high production costs. A drastic reduction
of production costs is, therefore, necessary to establish microbial surfactants as a general
alternative to conventional surfactants also outside of the previous market niches. There are
numerous books, reviews, and original papers covering near-exhaustive aspects of natural
surfactants and biosurfactants ranging from their application fields, microbial ecological,
biotechnological, to chemical structure analysis. A few of the selected books are those by Lang
and Trowitsch-Kienast (2002), Sen (2010), Soberón-Chávez (2010), and general reviews are
given by Satpute et al. (2010), Gutnick et al. (2011), and Merchant and Banat (2012). A thorough
review of the chemical structures in the broadest sense covering natural surfactants is given in a
series presented by Dembitsky (2004a,b, 2005a,b,c,d,e, 2006). This review focuses on low-
molecular weight microbial surfactants with a well-defined structure prepared by fermentation
covering the various types and classification of surfactants. The term biosurfactant is applied in
its strictest sense referring exclusively to surfactants taken directly from microbial sources,
without any organic synthesis. In addition to the fact that they can be produced by renewable
resources, biosurfactants are superior to their synthetic counterparts mainly by two essential
characteristics: their structural diversity and the specific biological activity of many structures. It
is evident that such additional properties exceeding pure reduction of surface tension make them
particularly interesting for some applications. Detailed consideration of the different
biosurfactants regarding their actual application capacity is not possible for most biosurfactants,
since the chemical structures and physical characterization of the surfactant properties alone are
no indication of the performance properties in product formulations. For this, the corresponding
biosurfactants must be available in quantities of about 0.1–1 kg. Thus, industrial product
development of biosurfactants is limited to some few biosurfactants, including spiculisporic acid,
sophorolipids, rhamnolipids, and mannosylerithritollipids. Below, the best-known biosurfactants
and, using some examples, the structural diversity and potential of microbial biosurfactants, in
general, are illustrated based on selected structures.

Unlike chemically synthesized surfactants, which are usually classified according to the nature of
their polar grouping, biosurfactants have been categorized mainly by their chemical composition
and microbial origin.[2]

The biosurfactants have been classified into low molecular mass molecules which efficiently
lower surface and interfacial tension; include glycolipids, lipopeptides and phospholipids and
high molecular mass polymers, which are more effective as emulsion stabilizing agents include
polymeric and particulate surfactants.[6] The major classes of biosurfactants include glycolipids,
lipopeptides, and lipoprotein, fatty acid, phospholipids, neutral lipids, and polymeric surfactants
(TableI).
Unlike chemically synthesized surfactants, which are usually classified to the nature of
their polar grouping, biosurfactants are generally categorized mainly by their chemical
composition and microbial origin. Rosenberg and Ron (1999) suggested that
biosurfactants can be divided into low molecular mass molecules which efficiently
lowers surface and interfacial tension, and high-molecular-mass polymers, which are
more effective than emulsion-stabilizing agent. These make up the two major classes.
The low-mass surfactants include glycolipids, lipopeptides and phospholipids, whereas
high-mass surfactants include polymeric and particulate surfactants. Most biosurfactants are
either anionic or neutral and the hydrophobic moiety is based on
long-chain fatty acids or fatty acid derivatives, whereas the hydrophilic portion can be a
carbohydrate amino acid, phosphate or cyclic peptide (Nitschke and Coast, 2007).
Table 1 shows the major biosurfactant classes and microorganisms that produce them.
Table 1: Major biosurfactant classes and microorganisms involved
Major groups and type of biosurfactants Microorganisms involved in production
Glycolipids
Rhamnolipids Pseudomonas aeruginosa
 Trehalolipids Rhodococcus erythropolis
Arthrobacter sp.
Sophorolipids Candida bombicola,
Candida apicola
Mannosylerthritol lipids Candida antartica
Lipopeptides
Surface/iturin/fengycin Bacillus subtilis

Viscosin Pseudomonas fluorescens

Lichenysin Bacillus licheniformis

Serrawettin Serratia marcescens

Phospholipid Acinetobacter species
Corynebacterium lepus
Surface-active antibiotics
Gramicidin Brevibacterium brevis

Polymixin Bacillus polmyxa

Antibiotic TA Myxococcus xanthus

Fatty acids/ neutral lipids Corynebacterium

insidibasseasum
Polymeric surfactants
Emulsan Acinetobacter

calcoaceticus RAG-1

(ATCC 31012)

Alasan Acinetobacter radioresistens

Liposan Candida lypolytica

Lipomanam Candida tropicalis

Particulate biosurfactants

Extracellular vesicles Acinetobacter calcoaceticusLFF

H01-N

Whole microbial cells Cyanobacteria

Source: Muthusamy, (2008).
There are many types of biosurfactants each produced by a specific microorganism.
The following are some of the various types of biosurfactants:
A). Glycolipids
Most known biosurfactants are glycolipids and are the most common types. They consist of
mono-, di-, tri-, and tetrasaccharides which include glucose, mannose, galactose, glucuronic acid,
rhamnose and galactose sulphate. The fatty acid component usually has a composition similar to
that of phospholipids of the same microorganism (Veenanadig et al., 2000; Chen et al., 2007).
Also, they are made up of carbohydrates in combination with long-chain aliphatic acids or
hydroxyaliphatic acids (Banat and Desai, 1997).The linkage is by means of either ether or an
ester group. Among the glycolipids, the best known are rhamnolipids, trehalolipids and
sophorolipids (Desai and Banat, 1997; Karanth et al., 1999) and the best-studied glycolipid
bioemulsifiers, rhamnolipids, trehalolipids and sophorolipids are disaccharides that are acylated
with long-chain fatty acids or hydroxyl fatty acids (Rosenberg and Ron, 1999).
i. Rhamnolipids Bacteria of the genus Pseudomonas are known to produce glycolipid surfactant
containing rhamnose and 3-hydroxy fatty acids (Lang and Wullbrandt, 1999; Rahman et al.,
2002). While the OH group of one of the acids is involved in glycosidic linkage with the
reducing end of the rhamnose disaccharide, OH group of the second acids is involved in ester
formation. Since one of the carboxylic acid is free, the rhamnolipids are anions above pH 4.0.
Rhamnolipids are reported to lower surface tension, emulsify CxHy and stimulate growth of
Pseudomonas on nhexadecane (Pruthi and Cameotra, 1997). The pure rhamnolipid lowered the
interfacial tension against n-hexadecane in water to about 1mN/m and had a critical micelle
concentration (CMC) of 10 to 30mg/l depending on the pH and salt conditions (Lang and
Wanger, 1988). Production of rhamnose containing glycolipids was first described in
Pseudomonas aeruginosa by Jarvis and Johnson and a wide study and report has shown that
rhamnolipids produced by Pseudomonas aeruginosa are a mixture of homologous species RL1
(RhC10C10), RL2 (RhC10), RL3 (Rh2C10C10), and RL4 (Rh2C10) (Syldatk and Wangner,
1987; Lang and Wagner, 1987; Rahman et al., 2002).
ii. Sophorolipids These are a group of biosurfactants produced by Torulopsis sp. Sophorolipids
(SLs) consist of a dimeric sugar (sophorose) and a hydroxyl fatty acid, linked by a β glycosidic
bond (Asmer et al., 1988). According to Hu and Ju (2001) there are two types of SLs namely, the
acidic (non-lactonic) SLs and the lactonic SLs. The hydroxyl fatty acid moiety of the acidic SLs
has a free carboxylic acid functional group whilst that of the lactonic SLs forms a macrocyclic
lactone ring with the 4”hydroxyl group of the sophorose by intramolecular esterificaion. Until
recently, lactonic SLs have been reported to have attracted more commercial and scientific
attention than their acidic counterparts. They have measurable biocide activity (Lang et al.,
1989), whilst the acetylated lactonic SLs have been applied in cosmetics as antidandruff,
bacteriostatic agents and deodorants (Mager et al., 1987).
iii. Trehalolipids Another group of glycolipids are the trehalolipids, the serpentine group seen in
many members of the genus Mycobacterium is due to the presence of trehalose esters on the cell
surface (Asselineau and Asselineau, 1978). Disaccharide trehalose linked at C-6 and C-6` to
mycolic acid is associated with most species of Mycobacterium, Norcardia and
Corynebacterium. Mycolic acids are long-chain, α-branched, β-hydroxy fatty acids. Trehalolipids
from different organisms differ in the size and structure of mycolic acid, the number of carbon
atoms and the degree of unsaturation (Desai and Banat, 1997). Trehalose lipids from
Rhodococcus erythropolis and Arthrobacter sp. were found to lower the surface and interfacial
tensions in culture broth from 25 to 40 and 1-5 mN m-1, respectively (Li et al., 1984).
B). Lipoproteins and Lipopeptides Lipopepetides called surfactin are produced by Bacillus sp.
containing seven amino acids bonded to a carboxyl and hydroxyl groups of a 14-carbon acid.
Surfactin just as any other biosurfactant reduces surface tension from 72 to 27 mN m-1 with
concentrations as low as 0.005%, making surfactin one of the most powerful biosurfactants
(Kakinuma et al., 1969). The cyclic lipopeptide surfactin produced by Bacillus subtilis ATCC
21332 is an example of one of the most powerful biosurfactants. Another important characteristic
of surfactin is its ability to lyse mammalian erythrocytes and to form spheroplasts (Bernheimer
and Avigad, 1970). This property is being used to detect surfactin production through haemolysis
on blood agar (Rahman and Gakpe, 2008).
C). Fatty acids, Phospholipids, and Neutral lipids Large quantities of fatty acids and
phospholipid surfactants are produced by several yeast and bacteria during growth on n-alkanes
(Cirigliano and Carman, 1984). The fatty acids produced from alkanes by microbial oxidations
have received maximum attention as surfactants. Besides the straight-chain acids,
microorganisms produce complex fatty acids containing OH groups and alkyl branches. The
balance between the hydrophilic and lipophilic is directly related to the length of the
hydrocarbon chain in their structures. Some of these complex acids, for example corynomucolic
acids, are surfactants (Copper et al., 1981; Macdonald et al., 1981; Kretschmer et al., 1982).
Phosphatidyl ethanolamine produced by Rhodococcus erythropolis grown on n-alkane causes a
lowering of interfacial tension between water and hexadecane to less than 1mN/m and critical
micelle concentration (CMC) of 30mg/l (Kretschmer and Wagner, 1982). The phospholipids are
the major components of microbial membranes. When certain CxHy degrading bacteria or yeast
are grown on alkane substrates, the level of phospholipids increases greatly. Phospholipids from
hexadecane grown on Acinetobacter species have potent surfactant properties. Phospholipids
produced by Thiobacillus have been reported to be responsible for wetting elemental sulpur,
which is necessary for growth (Kapelli and Fiechter, 1979).
D). Polymeric biosurfactants Emulsan, liposan, alasan, lipoman and other polysaccharide-protein
complexes are known to be the best studied polymeric biosurfactants (Desai and Banat, 1997).
(i) Emulsan is an effective emisifying agent for hydrocarbons in water (Zosim et al., 1982) even
at a concentration as low as 0.001 to 0.01%. The bacterium Acinetobacter calcoaceticus was
successfully used to clear a cargo compartment of an oil tanker during its ballast voyage
(Gutnick and Rosenberg, 1977; Rosenberg et al., 1979). The cleaning phenomenon was due to
the production of an extracellular, high molecular weight emulsifying factor (Rosenberg et al.,
1979), emulsan. Other Acinetobacter emulsifiers, such as 8 to 6 strains of A. calcoaceticus
produced high amounts of emulisifier following growth on ethanol medium. This extracellular
fraction is extremely active in breaking (de-emulsifying) kerosene/water emulsion stabilized by a
mixture of Tween 60 and Span 60.
(ii) Liposan: Liposan is an extracellular water-soluble emulsifier synthesized by Candida
lipolytica and is composed of 83% carbohydrate and 17% protein (Cirigliano and Carman,
1984) with the carbohydrate portion being a heteropolysaccharide consisting of glucose,
galactose, galactosamine and galactoronic acid. A CxHy degrading yeast, Endomycopsis
lipolytica YM, produced an unstable alkane-solubilizing factor. Torulopsis petrophilium
produced different types of surfactants depending on the growth medium. On water-soluble
substrates, the yeast produced glycolipids which were capable of stabilizing emulsions. When
glucose was the substrate, the yeast produced a potent emulsifier.
(iii) Surfactants from Pseudomonas PG1 are an extremely efficient hydrocarbon- solubilizing
bacterium. It utilizes a wide range of CxHy including gaseous volatile and liquid alkane, alkenes
and alkyl benzenes.
(iv) Bioflocculant and emulsan from the filamentous Cyanobacterium phormidium J-1; the
change in cell surface hydrophobicity of Cyanobacterium phormidium was correlated with the
production of an emulsifying agent, emulsan. The partially purified emulsan has a molecular
weight (MW) greater than 10,000 Da and contains carbohydrate, protein and fatty acid esters.
Addition of emulsan to adherent hydrophobic cells resulted in their becoming hydrophilic and
detached from hexadecane droplets or phenyl sepharose beads.
(v) Emulsifying protein (PA) from Pseudomonas aeruginosa: The bacterium P.aeruginosa has
been observed to excrete a protein emulsifier. The protein PA is produced from long-chain n-
alkanes, 1-hexadecane, and acetyl alcohol substrates; but not from glucose, glycerol or palmitic
acid. The protein has a MW of 14,000 Da and is rich in serine and threonine.
E). Particulate biosurfactants
Extracellular membrane vesicles partition hydrocarbons to form a micro emulsion, which plays
an important role in alkane uptake by microbial cells. Vesicles of Acinetobacter species strain
HO1-N with a diameter of 20-50nm and a buoyant density of 1.158 cubic g/cm are composed of
protein, phospholipids and lipopolysaccharide (Kappeli and Finnerty, 1979). Also, microbial
cells with high cell surface hydrophobicities are most hydrocarbon degrading microorganisms,
many non-hydrocarbon degraders, some species of Cyanobacterium and some pathogens have a
strong affinity for hydrocarbon-water and air-water interfaces. In such cases, the microbial cell
itself is a surfactant.
FIGURE 11.4 Répartition des producteurs de biosurfactants par genre
2.6. Extraction and characterization of biosurfactant
The biosurfactant was extracted by adjusting the pH of cell-free supernatant to 2.0 and incubated
overnight. The acidified precipitate was then extracted with chloroform: methanol (3:1) and the
supernatant was rotary evaporated to get the crude biosurfactant.
2.6.1. Characterization of biosurfactant 2.6.1.1. Drop collapse method. The culture broth was
centrifuged at 8000×g for 20minat 4°C, and the supernatant was collected. The test
forthepresenceofbiosurfactantinthecell-freesupernatantwascarried out by drop-collapse method
(Tugrul and Cansunar, 2005).
2.6.1.2. Emulsification index. The emulsifying activity of the biosurfactant was evaluated by
determining the emulsification index (E24).TheE24
ofthecrudebiosurfactantwasdeterminedbyadding2ml of coconut oil, kerosene, diesel, petrol, and
1ml of the extracted biosurfactant in a centrifuge tube, vortexed for 5min and allowed to stand
overnight (Guangming et al., 2005). The E24 was calculated as
=∗E Height of emulsion layer Height of total solution 10024
2.6.1.3. Surface tension reduction property of biosurfactant. The biosurfactant has the property of
reducing the surface tension of water. The biosurfactant containing fermentation broth was
centrifuged at 10,000rpm for 20min and then the surface tension of
thesupernatantwasdeterminedbytheringmethodusingatensiometer at room temperature (de Luna
et al., 2009).
2.6.1.4. FT-IR analysis of biosurfactant. To identify the functional groups present in the
biosurfactant, Fourier Transform Infrared Spectroscopy (FT-IR) analysis was done. One
milligram of crude biosurfactant was dried in a freeze dryer and grounded with 100mg of KBr
and then analyzed in the FT-IR (Agilent Technologies, Cary 600 Series), obtaining the spectrum
in transmission mode in the range of 400–4000cm−1 (Suganthi and Ramani, 2017).
2.6.1.5. Thin layer chromatography. The crude biosurfactant was spotted on silica gel plate and
separated using the solvent system containing isopropanol:water:ammonium hydroxide (80:11:9)
for protein and chloroform:water:methanol (65:10:25) for lipid. The developed plates were then
sprayed with 0.2% (w/v) ninhydrin in ethanol followed by heating at 110°C, to detect proteins,
and placed in iodine chamber to detect lipid moieties (Ramani and Sekaran, 2012).
2.6.1.6. Determination of amino acid composition of biosurfactant by HPLC. The biosurfactant
was hydrolyzed at 100°C for 20h with 1N HCl and neutralized with 1N NaOH. The amino acid
content in the crude biosurfactant was analyzed using Agilent D-7000 HPLC amino acid
analyzer (Deng et al., 2016).
2.6.1.7. SDS-PAGE for biosurfactant. The molecular weight of the protein in the biosurfactant
was determined by 12% SDS-PAGE.
2.7. Biodegradation of oil sludge in the liquid medium
Submerged culture system was used to biodegrade the oil sludge by microbial consortia. The
experiments were performed in triplicates using 250mL flasks containing 100ml of mineral salt
medium with 1% oil sludge as the carbon source. The microbial consortia were inoculated to it
and incubated at 37°C for 30 days. An intermittent inoculation of microbial consortia at an
interval of 4 days was done to
achieve the complete degradation of oil sludge. The flasks consisting of mineral salt medium and
oil sludge without microbial consortia served as the control. The biosurfactant production,
activity of lipase, catalase and oxidoreductase and TPH content were monitored every day.
2.8. Characterization of the treated oil sludge
2.8.1. Total petroleum hydrocarbon content analysis Total petroleum hydrocarbon (TPH) was
extracted by taking 1g of oil sludge (before and after biodegradation) in 100ml MSM medium
using hexane (1:1 ratio). The solvent layer containing the TPH portion was separated using the
separating funnel. Thisprocedure was repeated twice to remove any TPH present in the flask. The
hexane was evaporated using the rotary evaporator and the TPH was measured gravimetrically.
The experiment was carried out in triplicates. The percentage of TPH was calculated by
=
−
×
Hydrocarbon degradation Initial weight of hydrocarbon Final weight of hydrocarbon Initial
weight of hydrocarbon (%) 100
2.8.2. GC-MS analysis The hydrocarbons present in the initial and treated oil sludge was
determined by extracting the samples with an equal volume of chloroform and the organic phase
was evaporated to obtain the hydrocarbons. The obtained hydrocarbons were then subjected to
GC-MS (Agilent Technologies, USA, 7890B GC system coupled with 5977A MSD system)
which is equipped with HP_5MS 5% phenyl methyl silox column with dimensions
30m×250μm×0.25μm. Helium was used as carrier gas with a flow rate of 1.5mlmin−1, in a split
ratio of 100:1. The analysis was carried out at a temperature of 55°C for 2min and then increased
from 55°C to 300°C at 5°C/min and maintained at 300°C for 40min. The obtained MS spectra
were compared with the reference spectra present in the NIST.Lib.
2.8.3. NMR (1H) analysis 5-30mg of lyophilized samples were dissolved in 650μl of deuterated
chloroform and the degraded products were analyzed by NMR (500MHz Bruker Avance III)
(Taiwo and Otolorin, 2009).
S.H. Suganthi et al. Journal of Environmental Management 220 (2018) 87–95
89
2.8.4. Heavy metal analysis by inductively coupled plasma optical emission spectroscopy (ICP-
OES) One gram of untreated (control) and treated oil sludge was refluxed for 2hat 95°C with
concentrated HNO3 along with 2mL of water and 3mL of 30% H2O2. After completion of
H2O2 digestion, the samples were cooled and 10mL of concentrated HCl was added and the
samples were slowly heated to 95°C and the heating continued for 15min with a re flux. The
digested samples, after cooling were subjected to inductively coupled plasma optical emission
spectrometry (ICP-OES) (Zaleckas et al., 2012).

RESULTATS
3.3.2. Surface tension reducing capability of biosurfactant The surface tension was measured
with the help of tensiometer. Up to 48h, the production yield ofcrude biosurfactant was low.
During the periodof48–96h, productionwasexponential andreachedamaximum at 96h and
thereafter the levels seem to be maintained constant up to 240h. These findings are corroborated
with the results of surface tension reduction wherein the surface tension starts to decrease up to
96h and reaching a minimum value at 120h and the remained low till day 7. In addition, the
emulsification index (E24) increased in a time-dependent manner up to 96h, and it reaches its
maximum at 120h with 66% (Fig. 1a). Thereafter, no obvious variation in E24 values was
observed. On the whole, the decrease in surface tension and increase in emulsification activity
were almost consistent with the production of biosurfactant.
3.3.3. Emulsification activity of biosurfactant The emulsification activity ranged from 56 to 83%
(Fig. S4) for different hydrocarbons. The ability of the produced biosurfactants to emulsify
petrol anddiesel oil isan important feature forbioremediation applications. The produced
biosurfactants can be efficiently used in the detoxification of industrial effluents and in
bioremediation of soil polluted by hydrocarbons (Parthipan et al., 2017).
3.3.4. Thin layer chromatography and SDS-PAGE of crude biosurfactant The biosurfactant was
spotted on TLC sheet and showed a positive reaction with ninhydrin and iodine vapour,
indicating the presence of both protein and lipid moieties in the biosurfactant (Fig. 1b). The
crude biosurfactant failed to answer for phenyl sulphuric acid assay suggesting the absence of
carbohydrate molecule. These results suggest that the biosurfactant produced during the
breakdown of oil sludge is a lipoprotein. The crude biosurfactant was loaded onto SDS-PAGE
(Fig. 1c) and the molecular weight of the proteins present in the biosurfactants was checked and
found that they ranged between 53kDa and 78kDa. This result confirms the presence of protein
in the biosurfactant. Ramani et al. (2012) also reported their biosurfactant as lipoprotein
biosurfactant in the presence of slaughterhouse lipid waste. To date, this is the first report on the
production of lipoprotein biosurfactant by using petroleum tank bottom oil sludge.
3.3.5. FT-IR of crude biosurfactant The FT-IR spectrum (Fig. 1d) showed the peak
corresponding to amides at 3409.93cm−1, stretching mode of the CO-N bond at
1633.19cm−1 and the N-H bond combined with the C-N stretching mode at 1422.05cm−1. The
peak at 1758.14cm−1 corresponds to C=O stretching vibrations of ester group present in lipid
portion of biosurfactant.Thepeaksat3409.93cm−1 and1500–1650cm−1 arenot found in the FT-IR
spectra of rhamnolipid biosurfactant, that distinguishes them from rhamnolipid biosurfactant
(Ramani et al., 2012).
3.3.6. Amino acid composition of the biosurfactant
Themajoraminoacidspresentinthebiosurfactantareasparticacid, glutamic acid, asparagine, glycine,
alanine, threonine, phenylalanine, tyrosine and valine (Table S1). The presence of high
percentage of aspartic acid and glutamic acid indicates that the produced biosurfactant may be
anionic in nature.
3.4. Biodegradation of oil sludge
Fig. 2 illustrates the different enzyme profile and surface tension
reductionpropertyoveraperiodof30days.Theexperimentisdesigned in such a way that thelevel of
enzymes and biosurfactant areincreasing constantly to achieve the complete degradation of oil
sludge. Hence, an intermittent inoculation was made at an interval of 4 days to maintain the
enzymes and biosurfactant level constant. Each strain was grown in mineral salt medium
containing 1% oil sludge and 5% (w/v) inoculum of each of organisms was inoculated
intermittently every 4 days. The biodegradation of the oil sludge was determined by GC-MS and
NMR and the heavy metal removal by ICP-OES. From GC-MS and NMR it is observed that the
aliphatic hydrocarbons after treatment with the
microbial consortia resulted in the production of alcoholic intermediates and finally into
carboxylic acid. This could be due to the breakdown of the hydrocarbon by means of produced
microbial enzymes and biosurfactant (Abdel-Megeed et al., 2010).
3.5. Characterization of treated oil sludge
3.5.1. Total petroleum hydrocarbon content analysis The initial TPH content before treatment
was 14.8% (Fig. 3). TPH is extracted by liquid-liquid extraction with the help of hexane. The
solvent layer containing the whole TPH portion is separated, the solvent is evaporated and the
TPH is measured gravimetrically. The TPH content ofthe oilsludge was reducedto 0.6% onthe
28thday representing 96% of hydrocarbon have been reduced.
3.5.2. GC-MS analysis The GC-MS analyses of the oil sludge before and after treatment with
microbial consortia was shown in Fig. 4 (a–d) respectively. It is inferred from Fig. 4a–d that the
microbial consortia caused the degradation of hydrocarbons present in the oil sludge. In the
initial chromatogram (Fig. 4a), the peaks were numerous with higher abundance, whereas, after
treatment with microbial consortia, peak height