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Preethy Chandran et. al.

/ International Journal of Engineering Science and Technology


Vol. 2(12), 2010, 6942-6953

BIOSURFACTANT PRODUCTION AND


DIESEL OIL DEGRADATION BY YEAST
SPECIES TRICHOSPORON ASAHII
ISOLATED FROM PETROLEUM
HYDROCARBON CONTAMINATED SOIL
PREETHY CHANDRAN
Environmental Biotechnology Division, School of Biosciences and Technology
VIT University, Vellore, Tamil Nadu -632014

NILANJANA DAS*
Environmental Biotechnology Division, School of Biosciences and Technology
VIT University, Vellore, Tamil Nadu -632014
Abstract:
The yeast Trichosporon asahii, isolated from petroleum-contaminated soil in India was found to be the potent
producer of biosurfactant in mineral salt media containing diesel oil as the carbon source and found to be an efficient
degrader of diesel oil (95%) over a period of 10 days. The crude biosurfactant was purified using silica gel column
chromatography followed by dialysis .With the use of FTIR spectroscopy, in combination with GC-MS analysis,
chemical structures of the purified biosurfactant was identified as sophorolipid species. When compared to synthetic
surfactants, including Tween 80 and sodium dodecyl sulfate, the biosurfactant showed high physicochemical
properties in terms of the surface activities. Involvement of biosurfactant in physiological mechanism of diesel
adsorption on yeast cell surface was characterized based on zeta potential measurement. When diesel oil was
emulsified with biosurfactant, the surface charge of the diesel was modified resulting more adsorption of diesel on
yeast cell surface. Biosurfactant production by yeast species was monitored using SEM analysis.
Keywords: Biosurfactant; Diesel; Yeast species; Sophorolipid; Emulsification.
1. Introduction
Biosurfactants are polymers, totally or partially extracellular, amphipathic molecules containing polar and non polar
moieties which allow them to form micelles that accumulate at interphase between liquids of different polarities
such as water and oil (Desai and Banat, 1997) thereby reducing surface tension and facilitating hydrocarbon uptake
and emulsification (Thavasi et al., 2009). The superior properties of biosurfactants include high biodegradability,
low toxicity, ecological acceptability, and production from cheaper substrates (Nitschke et al., 1994). Other
advantages include selectivity and specific activity at extreme temperatures, pH and salinity (Velikonja et al., 1993).
Because of these properties, biosurfactants have potential application in food, pharmaceutical and cosmetic
industries (Desai and Banat, 1997).
Biosurfactants are widely used in hydrocarbon bioremediation field since they can enhance the growth on
hydrophobic surface and can increase the nutrient uptake of hydrophobic substrates thereby overcoming the poor
availability of hydrocarbon contaminants to microorganisms (Canet et al., 2001; Sotivora et al., 2009). Synthetic
surfactants currently used are toxic and hardly degraded by microorganisms causing damage to the environment.
These hazards caused by synthetic surfactants have drawn much attention to microbial biosurfactants (Kiran et al.,
2009).
Extracellular excretions of biosurfactants by bacteria have been reported by many scientists
(Pornsunthorntawee et al., 2008; Kumar et al., 2008; Thavasi et al., 2008). Among the bacteria, Pseudomonas sp.
are best known for degrading hydrocarbon and producing biosurfactants mainly rhamnolipid in nature (Rahman et
al., 2002).Whereas few reports are available with production and characterization of biosurfactants by yeast.

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Discovery of new biosurfactant producing strains and finding the optimum condition for biosurfactant
production are vital for effective bioremediation of soils and ground water contaminated with hydrocarbons. Since
the reports are very less in case of yeast species as potent producer of biosurfactant, present study was conducted
using yeast species Trichosporon asahii capable of producing biosurfactant in the presence of diesel. The
biosurfactant was purified, characterized and the structure was elucidated. The physicochemical properties were
investigated comparing with those of the synthetic surfactants including Tween 80 and SDS. Involvement of
biosurfactant in adsorption of diesel was also studied based on zeta potential measurement of surface charges.
2. MATERIALS AND METHODS

2.1. Diesel oil and other chemicals


Diesel oil used in this study was obtained from local petrol pump (Tamil Nadu, India) was filter sterilized and used
throughout the studies. All other chemicals used in the present study were of highest purity grade, produced by
Sigma-Aldrich (USA).
2. 2. Yeast strain and culture growth conditions
The yeast Trichosporon asahii, isolated from petroleum hydrocarbon contaminated soil in India was identified to the
species level by Viktek 2 Compact Yeast card reader with the software version V2C 03.01 from CFRD (Council for
Food Research and Development), Kerala, India. The isolated strain was maintained in YEPD (Yeast extract,
Peptone, Dextrose) agar slants at 28 C and subcultured every two weeks.
2. 3. Production and extraction of crude biosurfactant
Bushnell-Haas medium with diesel oil was used for optimization and production of biosurfactant by yeast species.
The yeast species was cultured at different temperatures (25 C to 55 C), substrate concentrations (0.5 % - 5 % v/v
of diesel) and pH (3.5-9.5). All the experiments were carried out in 250 ml conical flasks containing 50 ml mineral
salt medium (Bushnell-Haas medium containing KH2PO4 1 g l-1, K2HPO4 1 g l-1, NH4NO3 1 g l-1, FeCl3 0.05
g l-1 and MgSO4. 7H2O 0.2 g l-1). The culture was maintained in a water bath shaker at 120 rpm for a period of 14
days. The culture broth was centrifuged at 10, 000 rpm for 10 min and extracted with Chloroform and methanol
(2:1 v/v). The solvents were removed by rotary evaporation and the resultant residue obtained was crude
biosurfactant. Weight of the biosurfactant was expressed in terms of milligrams per milliliter (dry weight).
2. 4. Purification of biosurfactant
Purification of the biosurfactant was done following the method of Thavasi et al. (2009). The crude residue was
purified on a silica gel (60-120) mesh column eluting with a chloroform/methanol gradient ranging from 20:1 to 2:1,
collecting ten fractions. Then fractions eluted were pooled and the solvents were evaporated. The resulting residue
was dialyzed against distilled water and lyophilized following the method of Li et al., 1984.
2. 5. Physiochemical characterization of biosurfactant
To study the biosurfactant production and activity, the yeast was grown in Bushnell-Haas medium with diesel as
carbon source (2 % v/v) for a period of 14 days and culture was withdrawn intermittently to screen the biosurfactant
production. The physicochemical properties of the crude biosurfactant was investigated and compared with those of
two commercial surfactants, Sodium dodecyl sulphate (SDS) and Tween 80. SDS is a well known anionic surfactant
and Tween 80 is a non ionic surfactant and emulsifier derived from polyethoxylated sorbitan and oleic acid.
2. 6. Surface tension measurement
The surface tension of the aqueous solution (distilled water as control + distilled water and crude
extract/SDS/Tween 80 as test) was measured by using a du Nouy ring type tentiometer (Kruss, K10T). The surface
measurement was carried out at 25 1 C after dipping the platinum ring in the solution for a while in order to attain
equilibrium conditions. The measurement was repeated three times and an average value was obtained. The Critical

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micelle concentration (CMC) was then determined from the break point of the surface tension versus its log of bulk
concentration curve. For calibration of the instrument, the surface tension of the pure water was measured before
each set of experiment.
2. 7. Drop collapse test
Qualitative drop collapse test was performed following the protocol of Bodour and Maier. (1998). 2 l of diesel oil
was added to the 96 well microtitre plates. The plates were equilibrated for 1 h at 37 C and 5l of culture
supernatant was added to the surface of the oil. The shape of drop on oil surface was observed after 1min. The
culture supernatant that make the oil drop collapsed was indicated as positive result and that drops remain beaded
were scored as negative which was examined with distilled water as control.
2. 8. Oil displacement test
The oil displacement test is a method used to measure the diameter of the clear zone, which occurs after dropping a
surfactant-containing solution on an oil-water interphase. The binomial diameter allows an evaluation of the surface
tension reduction efficiency of a given biosurfactant. The oil displacement test was done by adding 50 ml of distilled
water to a petri dish with a diameter of 15 cm. After that 20 l diesel oil was dropped onto the surface of the water,
followed by the addition of 10 l of cell culture supernatant. The diameter and the clear halo visualized under visible
light were measured after 30 s (Rodrigues et al., 2006).
2. 9. Microbial adhesion to the hydrocarbon (MATH)
Cell surface hydrophobicity was assessed by microbial adhesion to the hydrocarbon method (MATH) according to
Rosenberg et al. (1980). Yeasts cells were harvested from 7 days and 14 days grown culture by centrifugation at
10,000 x g for 10 min at 4 C and washed twice with PUM buffer (buffer salt solution (pH 7.0) containing KH2PO4 7.26 g l-1, K2HPO4 - 19.7 g l-1, urea - 1.8 g l-1 and MgSO4. 7 H2O - 0.2 g l-1). The cells were again suspended in
PUM buffer to fit an optical density of ca.1.0 (A0). Optical density was measured at 600 nm on UV- Visible
Spectrophotometer (Shimadzu). Diesel (500l) was added to 5 ml of microbial suspension and vortexed for 2 min.
The optical density of aqueous phase was measured (A1) after 10 min. The degree of hydrophobicity was calculated
as [1-(A0-A1)/A0.100%]. The experiment was repeated thrice.
3. 0. Measurement of emulsification activity
Emulsification ability of the biosurfactant towards diesel as well as other hydrocarbons was studied. A mixture of 6
ml of studied hydrocarbons and 4 ml, 1 mg/ml of the biosurfactant/SDS and Tween 80 were vortexed at a high speed
for 2 min. The emulsion activity was investigated after 24 h and the emulsification index (E24) was calculated by
dividing the measured height of the emulsion layer by the total height of the mixture and multiplying by 100
(Cooper and Goldenberg, 1987).
3. 1. Stability testing
Stability studies were carried out using the cell-free broth obtained by centrifuging the cultures at 5,000 g for 20
min. The pH of the biosurfactant (4 ml) was adjusted to 2.0-10 using NaOH or HCl after which E24 was determined.
To test the heat stability of the biosurfactant, the broth was heated at 10100 C for 15 min, cooled at room
temperature and emulsification index (E24) was determined. The effect of NaCl on biosurfactant stability was also
assayed at different NaCl concentrations (010%, w/v).
3. 2. Biochemical characterization of biosurfactant
Chemical composition of the biosurfactant was analyzed following standard methods. Carbohydrate content of the
biosurfactant was determined by the anthrone reagent method using 620 nm (Spiro, 1966). D-glucose was used as
standard. Lipid content was estimated adopting the procedure of Folch et al. (1956). Protein content was determined
using bovine serum albumin as a standard following the method of Lowry et al. (1951).

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3. 3. Fourier transform infrared spectroscopy
One milligram of lyophilized purified biosurfactant was ground with 100 mg of KBr and pressed with 7, 500 kg for
30 s to obtain translucent pellets. The infrared spectra were recorded on Mattson 1, 000 FT-England FTIR system
within the range of 500-4000 cm-1 wave number. All measurements consisted of 500 scans, and KBr pellet was used
as background reference.
3. 4. Gas chromatography Mass spectrophotometry
The methyl ester derivates of the fatty acids were prepared by mixing the purified biosurfactant (10 mg) with 5%
HCl- methanol reagent (1 ml). After the reaction was quenched with water (1 ml), the methyl ester derivatives were
extracted with n-hexane and then injected (1 l) into a Hewlett Packard (HP, Wilmington, DE, USA) gas
chromatograph (GC) model 5890 Series II Plus equipped with a capillary inlet and an HP Mass Selective Detector
(MSD) model 5972 set to scan from m/z 45 to m/z 600 at a scan rate of 1.2 scans per second. The capillary column
used was an SP-2340 (60m x 0.25 mm) (Supelco, Bellefonte, PA, USA). The oven temperature was programmed
from 130 C to 230 C at 2 C min1. The temperature of the injector port was 230 C and the detector transfer line
temperature was 240 C. The carrier gas was He at a flow rate of 1 ml min-1 and a split ratio of 50:1.
3. 5. Measurement of zeta potential
Surface charges on the yeast cell surface, diesel and emulsified diesel with biosurfactant were measured at room
temperature at different pH (3.5-9.5) with a Brookhaven 90+ zeta analyzer (Brookhaven Instrument Corp., NY,
U.S.A.). Emulsified diesel was prepared by mixing three times for 3 min 97% distilled water and 1% v/v surfactant
and 2% diesel at a power level of +200 V by an ultrasonic generator (Sonics-Vibracell ultrasonic processor).The
stock emulsions were diluted 10 fold in phosphate (0.01 M, pH 7.5) buffer before use.
3. 6. Biosurfactant monitoring
Yeast cells grown in Bushnell haas medium with 2% diesel for 72 h were subjected to SEM (JOEL JSM 5600LV)
analysis to determine the biosurfactant production. Sample for SEM was prepared by dipping 1 cm2 coverslips in 1
ml of 72 h grown yeast cultures with 5 108 CFU ml-1 and placed for 90 min of adhesion phase at 37 C. The
coverslip was then washed with sterilized phosphate buffer saline (PBS) to remove loosely adherent cells. One
millilitre of Bushnell haas medium with diesel was added to the washed pieces and incubated at 37C for 72 h.
Again the coverslip was washed with phosphate buffer, dried and fixed with 3% gluteraldehyde and dehydrated with
a series of ethanol solutions (50, 75, 95, and 100%) and subjected to SEM analysis.
3.7. Diesel oil degradation
Fifty milliliters of Bushnell- Haas medium in 250-mL Erlenmeyer flask (pH 7.5), containing, 2% diesel (optimized
concentration, v/v data not shown) were inoculated with 2 ml of inoculum of 108 CFU/ ml and were incubated
aerobically at 35 C on a rotary shaker operated at 120 rpm for 10 days. A control was incubated with heat killed
yeast species to monitor abiotic losses of the diesel substrate. Samples were withdrawn at specified intervals for
determining pH. At the end of the experiment, the residual diesel fuel in the entire flask was extracted using hexane
after adjusting the pH to 1 using 1N HCl. The degraded diesel was analyzed using Gas chromatography Mass
spectrophotometer (Obayori et al., 2009).
The degradation of diesel as whole was expressed as the percentage of diesel degraded in relation to the
amount of the remaining fractions in the appropriate abiotic control samples. The biodegradation efficiency (BE),
based on the decrease in the total concentration of hydrocarbons, was calculated by using the expression described
by Michaud et al., 2004:

As 100
100

Aac

Where As = total area of peaks in each sample, Aac = total area of peaks in the appropriate abiotic control. BE (%) =
Biodegradation efficiency.

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3. 8. Statistical analysis
The experimental data are presented in terms of arithmetic averages of at least three replicates and the standard
deviations are indicated by error bars.
4. Results and discussion
4. 1. Production and optimization of crude biosurfactant
Biosurfactant production by Trichosporon asahii on diesel oil was detected maximum at pH 7.5, at a temperature of
35 C and substrate concentration of 2% (v/v) diesel substrate (Fig. 1). Biosurfactant production was found to be
maximum at the early stationary phase on 10th day at all culture conditions tested. Higher concentration of
biosurfactant at the early stationary phase may be due to the release of cell-bound biosurfactant into the culture broth
which led to the raise in extracellular biosurfactant concentration (Goldman et al., 1982).

Fig. 1. Biosurfactant production by Trichosporon asahii at different pH (B1), at different temperature (B2), at different diesel concentrations (B3)
in Bushnell- Hass medium over a period of 14 days.

4. 2. Physiochemical characterization of biosurfactant


The drop collapse test and oil displacement tests are indicative of the surface and wetting activities (Youssef et al.,
2004). The oil displacement test is an indirect measurement of surface activity of a surfactant sample tested against
oil; a larger diameter represents a higher surface activity of the testing solution (Rodrigues et al., 2006). In the
present study surface activities of the crude biosurfactant was investigated in comparison with that of Tween 80 and
SDS. Drop collapse test and oil displacement test were highly positive for crude biosurfactant of Trichosporon
asahii than commercial surfactants, Tween 80 and SDS, which indicated high surface activity (Table 1).
Table 1. Physicochemical characterization of biosurfactant produced by Trichosporon asahii in Bushnell- Haas medium supplemented with 2%
diesel after 7 days of cultivation

Characterization

Surface tension
(mN/m)

CMC
(mg/l)

Drop collapse test

Oil displacement
(cm2)

Distilled water

72 0.5

Negative

Negative

Negative

Crude extract a

30 0.6

197

117 1.5

SDS

34 0.5

380

88 0.7

Tween 80

37 0.8

1250

78 0.9

+++
c

++
++

All values are means SD for triplicate cultures


Crude extract 1a crude form of biosurfactant from Trichosporon asahii
b
+ + + indicate highly positive drop collapse test
c
+ + indicate moderate positive drop collapse test

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Emulsification index (%)

The ability of the crude biosurfactant as well as Tween 80 and SDS to reduce the surface tension of distilled water
was compared (Table 1). In case of biosurfactant produced by Trichosporon asahii reduced the surface tension of
distilled water to a minimum value with low value of CMC. As shown in Table 1 Tween 80 and SDS were found to
reduce the surface tension but with high CMC values. The results suggested that the biosurfactant from the yeast
species provided excellent properties in terms of reduction of surface tension and a low value of CMC.
Emulsification index of the biosurfactant from Trichosporon asahii was measured as 89 0.7 % over a
period of 7 days. Whereas emulsification index of the commercial surfactants, Tween 80 and SDS was found to be
67 0.9 and 78 0.8%. Thus the present study showed that the surface activities of biosurfactant from yeast species
was quite high than the commercial surfactants. Biosurfactants could effectively emulsify and stabilize emulsions
with diesel as well as various types of hydrocarbon substrates (Fig. 2). Biosurfactant was found to emulsify
tetracosane (C24), long chain hydrocarbon effectively. Though commercial biosurfactants like Tween 80 and SDS
could emulsify the tested hydrocarbons viz. hexadecane, pentadecane, dodecane, hexane, octane and pentane but
failed to emulsify the longer chain hydrocarbon like tetracosane. Trichosporon asahii showed high cell surface
hydrophobicity over a period of 7 days (92 0.9 %) and almost constant for a period of 14 days (90 0.8 %). The
cell surface hydrophobicity was related to the biosurfactant secreted on the cell surface, helping adhesion of
microorganisms to the hydrocarbons, and resulting in the effective degradation (Rosenberg et al., 1980; Maneerat,
2005). A positive correlation ( = 0.986) was noted between cell surface hydrophobicity and emulsification index of
the biosurfactant. From the point of view of microbial degradation, dissolution and emulsification of hydrocarbons
appeared to have a positive effect on degradation rate (Amund and Adebiyi, 1991). Biosurfactant emulsion with
diesel oil was found to be stable for more than one month at room temperature without any change in emulsification
activity.

100
90
80
70
60
50
40
30
20
10
0

BS1
SDS

xa

sa
n

ne

te

tr a

co

he

pe

nt

an

ne
ta

e
oc

ca
n
de
do

ad
nt
pe

he

xa

de

ca

ec
an

ne

Tw een 80

Hydrocarbon substrates

Fig. 2. Emulsification activity (E24) of the crude biosurfactant produced by Trichosporon asahii (BS1) towards different hydrocarbon substrates
compared with Tween 80 and SDS.

The emulsification index values of biosurfactant were also measured at different temperatures, pH and
NaCl concentrations (Fig 3). Biosurfactant isolated from Trichosporon asahii showed emulsification activity at
temperature of 10 -100 C and pH ranges from 2 to 8. Less emulsification activity was retained at pH 10. The
retention of over 60% emulsification activity at 10 C and 100 C suggested that the biosurfactant produced by
Trichosporon asahii might be useful in extreme environments such as temperate marine compartments and
industrial systems where extremes of temperature are integral elements. Biosurfactant might also be useful in acidic
and alkaline environment conditions since it can retain its activity over a pH ranges from 2 to 10. Emulsification
index of the biosurfactant produced were found to be inversely proportional to the NaCl concentration, but still
retained its activity at high concentration of NaCl (8-10%). This suggested that the biosurfactant might be useful in
marine environments and other systems where salt concentration is above physiological level. There are reports on
some biosurfactants produced by bacteria showing stability in presence of high salt concentration (Obayori et al.,
2009; Sarubbo et al., 2007).

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Fig. 3. Effect of pH (a), temperature (b), NaCl concentration (c) on the emulsification activity of biosurfactants produced by Trichosporon asahii
grown on diesel

4. 3. Biochemical and structural characterization of biosurfactant


Carbohydrate, Protein and lipid estimation of the biosurfactant produced by yeast species, Trichosporon asahii was
classified as glycolipids with carbohydrate and lipid as major constituents (Table 2).
Table 2. Biochemical characterization of biosurfactant produced by Trichosporon asahii

Chemical analysis

Results (%)

Carbohydrate

31.85 0.8

Lipid

68.20 0.7

Protein

Nil

Molecular compositions of biosurfactant were evaluated by FT-IR. Fig. 4 represents the spectra of the purified
freeze dried biosurfactant from Trichosporon asahii. The broad band observed in biosurfactant was at 3434 cm-1
corresponds to the O-H stretch. The asymmetrical stretching (Vas CH2) of methylene occur at 2926 cm-1. The band
1625 cm-1 is from stretching of unsaturated CC bonds. Lactones and esters have two strong absorption bands
arising from CO and CO stretching. The CO absorption band at 1744 cm-1 includes contributions from that of
lactones, esters, or acids. The stretch of CO band of C (O) OC in lactones appears at 1157 cm-1. The sugar
CO stretch of COH groups is at 1048 cm-1. From the FT-IR data it is evident that lactonic form of biosurfactant
is dominant in the yeast species.

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Fig. 4. FT-IR spectra of purified biosurfactant produced by Trichosporon asahii.

GC-MS analysis of purified biosurfactant was done to elucidate structures and an MS spectrum of biosurfactant as
shown in Fig. 5. The significant ions occurred at m/z 662 mass spectra of biosurfactant from Trichosporon asahii
and the corresponding chemical structure was determined as diacetate lactonic sophorolipids with fatty acid moiety
C18:1. Trichosporon asahii was found to be the potent producer of sophorolipids which was in diacetate lactonic
form. Since sophorolipids are useful in oil recovery, for removal of hydrocarbons from soils and groundwater tables
(Pesce, 2002; Ducreux et al., 1997), the sophorolipid produced by the yeast species will definitely show the potential
application in all the fields mentioned above.

Fig. 5. Mass spectrum of purified biosurfactant (sophorolipid) produced by Trichosporon asahii grown on diesel using silica column
chromatography. Mass spectrum represents diacetate lactonic sophorolipid (MW 662) produced by Trichosporon asahii.

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4. 4. Surface charge measurement
The zeta potentials of the yeast species Trichosporon asahii, biosurfactant emulsified diesel and diesel emulsion at
different pH are shown in Fig. 6. Mostly all the microbial cells are negatively charged at neutral pH. Here the yeast
species exhibited negative zeta potential in all the pH ranges. The biosurfactant emulsified diesel showed highly
positive zeta potential up to pH 7.5 whereas the zeta potential turned to less positive when pH is above 7.5.
Therefore, the maximum degradation can be observed up to pH 7.5, due to the attachment of highly positive charge
biosurfactant emulsified diesel to the negative charge yeast cell surface. Whereas diesel emulsion showed negative
charge in all the pH, so there will be weak attachment of diesel to yeast cell surface. These results showed that
biosurfactant is involved in the surface charge modification of diesel and enhancing the adsorption of diesel to yeast
cell surface.

Zeta potential, mV

20
10
Y4
B4

0
0

10

-10
-20
-30
-40

pH

Fig. 6. Zeta potential and electrostatic interaction between biosurfactant emulsified diesel (B4), Diesel emulsion (D), Trichosporon asahii (Y4) at
different pH.

4. 5. SEM analysis for biosurfactant production


The biosurfactant or exopolymer production by the yeast species, Trichosporon asahii was well observed in SEM
photograph (Fig. 7). Yeast cells with diesel oil resulted the formation of sticky colourless matrix of exopolymers
interconnecting individual cells into an intricate network of coherent mass after 72 hours of incubation
Microencapsulation of yeast cells in their exopolymers was also noted in SEM image.

Colourless sticky matrix of


exopolymer (biosurfactant)

Fig. 7. SEM image showing exopolymer production (sticky colorless matrix) by Trichosporon asahii grown in Bushnell haas medium containing
diesel after 72 h of incubation.

4. 6. Diesel oil degradation


In GC-MS analysis, the control system consists of n-alkanes (C9-C26), branched alkanes, naphthalene derivatives,
substituted naphthalenes and isoprenoid alkanes (pristine, phytane) (Fig 8a), where as in test system the yeast
species degraded almost all alkanes (Fig 8b). Branched chain alkanes and naphthalene derivates were also reduced.

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Percentage degradation of individual compounds was represented in Table 3. The GC-MS analysis suggested that
yeast species degraded almost all the hydrocarbon present in diesel (C9 C26), even the isoprenoid compounds like
pristine and phytane. Overall percentage degradation of compounds in diesel by Trichosporon asahii was found to
be 95.01%. Hence these yeast species can be considered as suitable bioremediation agent for the recuperation of
areas contaminated by diesel oil as well as other hydrocarbons because diesel oil is a model hydrocarbon to study
the hydrocarbon degradation since it consists of a variety of molecules such as paraffin, olefins, naphthalene and
aromatic compounds (Ilori et al., 2008).

Figure 8. GC-MS profiles of diesel oil extracted from aqueous phase of Bushnell hass medium after 10 days of incubation (a) GC-MS profiles of
diesel inoculated with heat killed yeast species (b) Inoculated with Trichosporon asahii after incubation period of 10 days. n-alkanes number
designates the number of carbon atoms; Br branched alkanes; Na substituted naphthalenes; Fa Farnithane compounds; Pr pristane; Ph phytane.
The compounds were identified by comparing of their retention times and mass spectra with authentic standards also confirmed by Wiley mass
spectra library.

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Table 3. Percentage of biodegradation efficiency of individual hydrocarbon compounds in diesel by Trichosporon asahii in Bushnell- Haas
medium after 10 days of incubation.

Compounds
C9
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
C25
C26
Naphthalene derivatives
compound 1
Naphthalene derivatives
compound II
Branched alkanes compound 1
Branched alkanes compound II
Phytane compound 1
Pristane compound 1
Pristane compound II
Farnithine compound I
Farnithine compound II
BEa

Trichosporon asahii
BEa (%)
100
100
98.9
98
97.2
96.1
95.2
94.1
93.2
93.1
92.8
92.5
92.3
92.1
91.9
91.9
91.7
98
95.5
96.1
94.5
94.1
94
93.2
95.2
94.5
92.2

- Biodegradation efficiency

5. Conclusions
The yeast species Trichosporon asahii isolated from hydrocarbon contaminated sites was found to be the potent
producer of biosurfactant identified as sophorolipid as well as efficient degrader of diesel oil. The biosurfactant
having higher hydrophobicity, emulsification activity, surface tension reduction and wide range of hydrocarbon
emulsification activity, can be feasibly used towards in situ bioremediation of ground water. Stability of the
biosurfactant in wide range of pH, temperature and salinity also can enable this compound for use under extreme
environmental conditions.
Acknowledgment
Authors of this article would like to thank VIT University for providing Lab facility and financial support for the
smooth conduct of the work.
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