Lipopeptide a biosurfactant

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.

ISSN: 0975-5462 6942


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

ISSN: 0975-5462 6943


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).

ISSN: 0975-5462 6944


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:

Aac

As 100100

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

ISSN: 0975-5462 6945


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)

Oil displacement (cm2)

Drop collapse test

Distilled water 72 0.5 Negative Negative Negative

Crude extract a 30 0.6 197 117 1.5 b + + +

SDS 34 0.5 380 88 0.7 c+ +

Tween 80 37 0.8 1250 78 0.9 + +

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

ISSN: 0975-5462 6946


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.

0102030405060708090

100

hexa

deca

ne

penta

deca

ne

dode

cane

octan

e

penta

ne

hexa

ne

tetrac

osan

e

Hydrocarbon substrates

Emul

sific

atio

n in

dex

(%)

BS1

SDS

Tw een 80

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).

ISSN: 0975-5462 6947


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.

ISSN: 0975-5462 6948


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.

ISSN: 0975-5462 6949


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.

-40

-30

-20

-10

0

10

20

0 2 4 6 8 10

pH

Zeta

pot

entia

l, m

V

Y4B4D

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.

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.

Colourless sticky matrix of exopolymer (biosurfactant)

ISSN: 0975-5462 6950


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.

ISSN: 0975-5462 6951


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 Trichosporon asahii

BEa (%) C 9 100 C10 100 C11 98.9 C12 98 C13 97.2 C14 96.1 C15 95.2 C16 94.1 C17 93.2 C18 93.1 C19 92.8 C20 92.5 C21 92.3 C22 92.1 C23 91.9 C24 91.9 C25 91.7 C26 98 Naphthalene derivatives compound 1

95.5

Naphthalene derivatives compound II

96.1

Branched alkanes compound 1 94.5 Branched alkanes compound II 94.1 Phytane compound 1 94 Pristane compound 1 93.2 Pristane compound II 95.2 Farnithine compound I 94.5 Farnithine compound II 92.2 BEa - 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. References [1] Amund, O.O.; Adebiyi, A.G. (1991). Effect of viscosity on the biodegradability of automotive lubricating oils. Tribol. Intern., 24(4), 235 -

237.

ISSN: 0975-5462 6952


[2] Bodour, A.A.; Maier, R.M. (1998). Application of a modified drop collapse technique for biosurfactant quantification and screening of biosurfactant producing microorganisms. J. Microbiol. Methods., 32(3), 273 - 280.

[3] Canet, R.; Birnstingl, J.G.; Malcolm, D.G.; Lopez-Real, J.M.; Beck, A.J. (2001). Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by native microflora and combination of white rot fungi in coal-tar contaminated soil. Biores. Technol., 76(2), 113 - 117.

[4] Cooper, D.G.; Goldenberg, B.G. (1987). Surface active agents from two Bacillus species. Appl. Environ. Microbiol., 53(2), 224 - 229. [5] Desai, J.D.; Banat, I.M. (1997). Microbial production of biosurfactants and their commercial potentials. Microbiol. Mol. Biol. Rev., 61(1),

47 - 64. [6] Ducreux, J; Ballerini, D.; Baviere, M.; Bocard, C.; Monin, N. (1997). Composition containing a surface active compound and glycolipids

and decontamination process for a porous medium polluted by hydrocarbons. US patent 5654192. [7] Folch, J.M.; Lees, M.; Stanly, H.S. (1956). A simple method for the isolation and quantification of total lipids from animal tissues. J. Biol.

Chem., 226, 497 - 509. [8] Goldman, S.; Shabtai, Y.; Rubinovitz, C.; Rosenberg, E.; Gutnick, D.L. (1982). Emulsan in Acinetobacter calcoaceticus RAG-1:

distribution of cell free and cell associated cross reacting materials. Appl. Environ. Microbiol., 44(1), 165 - 170. [9] Ilori, M.O.; Adebusoye, S.A.; Ojo, A.C. (2008). Isolation and characterization of hydrocarbon degrading and biosurfactant producing yeast

strains obtained from lagoon water. World. J. Microbiol. Biotechnol., 24, 2539-2545. [10] Kiran, G.S.; Hema, T.A.; Gandhimathi, R.; Joseph Selvin; Anto Thomas, T.; Rajeetha Ravji, T.; Natarajaseenivasana, K. (2009).

Optimization and production of a biosurfactant from sponge associated marine fungus Aspergillus ustus MSF3. Colloids. Surfurce. B., 73(2), 250 - 256.

[11] Kumar, M.; Leon, V.; Materano, A.D.S.; Joseph Selvin; Anto Thomas T, Rajeetha Ravji T, Natarajaseenivasan, K. (2008). Biosurfactant production and hydrocarbon degradation by halotolerant and thermotolerant Pseudomonas sp. World. J. Microbiol. Biotechnol., 24(7), 1047 - 1057.

[12] Li, Z.Y.; Wagner, F.; Witte, L.; Wary, V. (1984). Formation and identification of interfacial active glycolipids from resting microbial cells. Appl. Environ. Microbiol., 48(3), 610 - 617.

[13] Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem., 193, 265 - 75.

[14] Maneerat, S. (2005). Biosurfactants from Marine Microorganisms. J. Sci. Technol., 27, 1263. [15] Michaud, L.; Lo Giudice, A.; Saitta, M.; De Domenico Vivia, M. (2004). The biodegradation efficiency on diesel oil by two psychrotrophic

Antarctic marine bacteria during a two-month-long experiment. Mar. Res. Bull., 49, 405409. [16] Nitschke, M.; Pastore, G.M. (1994). Biosurfactant production by Bacillus subtilis using cassava processing effluent. Appl. Biochem.

Biotechnol., 112(3), 163 - 172. [17] Obayori, O.S.; Ilori, M.O.; Adebusoye, S.A.; Oyetibo, G.O.; Amund, O.O. (2009). Degradation of hydrocarbons and biosurfactant

production by Pseudomonas sp strain LP1. World. J. Microbiol. Biotechnol., 25(9), 1615-1623. [18] Pesce, L. (2002). A biotechnological method for the regeneration of hydrocarbons from degs and muds, on the base of biosurfactants. World

patent 02/062495. [19] Pornsunthorntawee, O.; Wongpanit, P.; Chavadej, S.; Abe, M.; Rujiravanit, R. (2008). Structural and physiochemical characterization of

crude biosurfactant produced by Pseudomonas aeruginoa SP4 isolated from petroleum-contaminated soil. Biores. Technol., 99, 1589 - 1595.

[20] Rahman, K.S.M.; Banat, I.M.; Thahira, J.; Thayumanavan, T.; Lakshmanaperumalsamy, P. (2002). Bioremediation of gasoline contaminated soil by a bacterial consortium amended with poultry litter, coir pith, and rhamnolipid biosurfactant. Biores. Technol., 81(1), 25 - 32.

[21] Rodrigues, L.R.; Teixeira, J.A.; Van der Mei, H.C.; Oliveira, R. (2006).Physiochemical and functional characterization of a biosurfactant produced by Lactococcus lactis 53. Colloid.Surface. B., 49(1), 79 - 86.

[22] Rosenberg, M.; Gutnick, D.; Rosenberg, E. (1980). Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS. Microbiol. Lett., 9(1), 29 - 33.

[23] Sarubbo, L.A.; Farias, C.B.B.; Campos-Takki, G.M. (2007). Co-utilization of canola oil and glucose on the production of a biosurfactant by Candida lipolytica. Curr. Microbiol., 54(1), 68-73.

[24] Sotivora, A.; Spasova, D.; Vasileva-Tonkova, E.; Galabova, D. (2009). Effect of rhamnolipid biosurfactant on cell surface of Pseudomonas aeruginosa. Microbiol. Res., 164(3), 297 - 303.

[25] Spiro, R.G. (1966). Analysis of sugar found in glycoproteins. Methods. Enzymol., 8, 7 - 9. [26] Thavasi, R.; Jayalakshmi, S.; Balasubramanian, T.; Ibrahim, M.B. (2008). Production and characterization of a glycolipid biosurfactant

from Bacillus megaterium using economically cheaper sources. World. J. Microbiol. Biotechnol., 24(7), 917 - 925. [27] Thavasi, R.; Subramanyam Nambaru, M.; Jayalakshmi, S.; Balasubramanian, T.; Ibrahim, M.B. (2009). Biosurfactant production by

Azotobacter chroococcum isolated from the marine environment. Mar. Biotechnol., 11(5), 551 - 556. [28] Velikonja, J.; Kosaric, N. Biosurfactant in food application. (1993). In Biosurfactants: production, properties applications; Kosaric, N., Eds.;

New York, Marcel Dekker. 419 - 446. [29] Youssef, N.H.; Duncan, K.E.; Nagle, D.P.; Savage, K.N.; Knapp, R.M.; Michael, J.M. (2004). Comparison of methods to detect

biosurfactant production by diverse microorganisms. J. Microbiol. Methods., 56(3), 339 - 347.

ISSN: 0975-5462 6953

Date post:	18-Dec-2015
Category:	Documents
Upload:	parthuz
View:	28 times
Download:	4 times

Lipopeptide a biosurfactant

Documents