IDENTIFICATION OF PSEUDOZYMA HUBEIENSIS Y10BS025 AS A POTENT PRODUCER OF GLYCOLIPID BIOSURFACTANT MANNOSYLERYTHRITOL LIPIDS

Mannosylerythritol Lipids (MEL’s) are glycolipid biosurfactants that contain 4-O-β-D-mannopyranosylmeso-erythritol as a hydrophilic moiety and fatty a cids as a hydrophobic moiety. MEL’s are abundantly produced by several kinds of microorganism and are on of the most promising biosurfactants currently known. The search for a novel endogenous producer o f MEL’s was undertaken based on the available collection of the yeast strains from the genus Pseudozyma. Using thin layer chromatography and based on morphological and molecular taxonomic analysis usin g the D1/D2 domains of the large subunit 26S rRNA gene, Pseudozyma hubeiensis Y10BS025 was found to be a potential producer of ME L’s from soybean oil. The structure of the major glycolipid produced by t he strain was analyzed by H and C nuclear magnetic resonance and was found to be similar to those of w ell known MEL-A, -B and -C respectively. Under improved shaking culture conditions, using yeast ex tract as nitrogen source and soybean oil as substra te, a maximum yield of 115±3.2 g.L −1 of MEL’s for 8 days of fermentation was achieved. The major fatty acids of MEL’s produced by P. hubeiensis Y10BS025 were C-18 acids, considerably different f rom those of MEL-C produced by other Pseudozyma strains such as P. antarctica and P. shanxiensis. The main product, MEL-C produced by P. hubeiensis Y10BS025 exhibited surface-tension-lowering activi ty. The results demonstrated that the newly isolated P. hubeiensis Y10BS025 provided high efficiency in MEL’s production and would thus be highly advantageous in commercial production of promising biosurfactants.


INTRODUCTION
Biosurfactants are extracellular amphipathic compounds produced by a variety of microorganisms and have numerous applications in the food and pharmaceutical industries, as well as in environmental protection and energy-saving technology (Morita et al., 2009a;Bhangale et al., 2013). The application of biosurfactants in cosmetics has also been addressed (Morita et al., 2010;Yamamoto et al., 2012;Morita et al., 2013). In addition, biosurfactants are considered to play important roles in differentiation induction with respect to human leukemia, rat pheochromocytoma and mouse melanoma cells, as well as high affinity binding toward different immunoglobulins and lectins (Morita et al., 2009b).
In recent years, biosufactants have attracted considerable interest due to their biodegradability, mild production conditions and a variety of functions Science Publications AJBB (Liu et al., 2011). However, the relatively low efficiency in their production and recovery has limited wide industrial use. It is therefore considered important to search for novel biosurfactant producers and to improve the systems for large-scale production of biosurfactants. Based on the structure of their hydrophilic moiety, biosurfactants are categorized into four major groups; glycolipid type, amino acid type, carboxylic acid type and polymer type (Morita et al., 2009a). One of the most promising glycolipid biosurfactants are the Mannosylerythritol Lipids (MEL's). MEL's contain 4-O-β-D-mannopyranosylmeso-erythritol as a hydrophilic moiety and fatty acids as a hydrophobic moiety (Onghena et al., 2011;Morita et al., 2011).
MEL's are abundantly produced by several kinds of microorganisms (Morita et al., 2009a). Previously, a number of yeast strains have been reported to secrete MEL's in a large amounts. These include P. aphidis (Onghena et al., 2011), P. antarctica (Bhangale et al., 2013), P. parantarctica (Morita et al., 2009b), P. siamensis, P. graminicola, P. tsukubaensis, U. cynodontis (Morita et al., 2009a), U. scitaminea (Morita et al., 2011), U. maydis (Liu et al., 2011) and P. hubeiensis SY62 (Konishi et al., 2013). The MEL's produced by Pseudozyma antarctica (ATCC 32657) are consisted of three major components, MEL-A, MEL-B and MEL-C. In addition, the structure can vary in the number of carbon atoms and unsaturation in fatty acid moiety (Bhangale et al., 2013). The aim of the present study, was to investigate for the first time, the production of MEL's using yeast strain P. hubeiensis Y10BS025 isolated in Indonesia. During the course of our study, P. hubeiensis Y10BS025, (a recently identified strain) was found to secrete a more hydrophilic glycolipids than the known MEL's. Here we describe the purification and structural analysis of MEL's produced with a varied ratio of glucose and glycerol as carbon sources. We also analyze the fatty acid composition and the surfactant activity of these glycolipids MEL's.

Yeast Strain and Culture
An endophytic yeast (isolated from sirih leaf, Piper betle L.), Pseudozyma Y10BS025 was obtained from the Biotechnology Culture Collections (BTCC), Indonesian Institute of Sciences (LIPI). Cultivation of yeast seed culture was started by inoculating yeast cells grown on slants into a 100 mL flask containing 20 mL growth basal medium (4% w/v glucose, 0.3% w/v NaNO 3 , 0.03% w/v MgSO 4 , 0.03% w/v KH 2 PO 4 and 0.1% w/v yeast extract) at 30°C on a rotary shaker (200 rpm) for 2 days. The yeast inoculums (10%, v/v) was added to the 200 mL production basal medium containing 8% v/v soybean oil and cultivation was performed by the submerged method with shaking for 8 days.

Molecular Phylogenetic Analysis
The genomic DNA of the strain of Pseudozyma Y10BS025 was prepared with a genomic DNA isolation kit (Go Beads, Japan) after cell rupture by vortexing for 3 min and heating it at 98°C for 10 min. The D1/D2 domain of the large ribosomal subunit (26S rRNA) gene was sequenced directly from PCR products generated using the primer NL-1 (5'-GCATATCAATAAGCGGAAAAG)-F and NL-4 (5'GGTCCGTGTTTCAAGACGG)-R.
The D1/D2 sequences of related taxa were retrieved from GenBank. The BLAST program was used for similarity search in the database available on the National Centre Biotechnology Information (NCBI). Multiple alignment was performed with the CLUSTAL W program. Phylogenetic analysis was performed using the neighbor-joining method with the program MEGA 5 (Tamura et al., 2011).

Isolation and Purification of Glycolipid MEL's
After cultivation, the post culture was harvested by centrifugation at 10000×g for 20 min at 10°C and the resulting supernatant, including glycolipid biosurfactant, was extracted using ethylacetate in 1:1(v:v) for 1 h (Morita et al., 2011). The organic layer (top phase) was separated from the water layer (bottom phase). Its volume was measured and concentrated using vacuum evaporation. The stepwise conventional extraction of the organic phase using different solvents was used to purify biosurfactant MEL's. A complete separation of residual soybean oil and fatty acids was achieved by using nhexane, methanol and water (1:6:3, v/v) as solvent mixture with subsequent threefold extraction by n-hexane. The lyophilized aqueous phase resulted in a MEL's fraction.
The pure MEL's were analyzed by thin layer chromatography on a silica gel plate G-60 (Merck). TLC was performed with chloroform-methanol-NH 4 OH (65:15:2, v:v:v) as the solvent system.

AJBB
Chromatograms were stained in the anthrone reagents (2%, w/v) in concentrated H 2 SO 4 and by heating at 115°C for 5 min for visualization (blue spots). Purified MEL-A, MEL-B and MEL-C, prepared as reported previously, was used in the following experiments as the standards (Morita et al., 2011).

Optimation of MEL's Production
In order to determine optimum medium formulation for MEL's production, the yeast strain was grown on minimum basal medium with varied ratios of glucose and glycerol as the carbon source and with different vegetable oils as an inducer. The ratios of glucose: Glycerol tested were (100:0, 75:25, 50:50, 25:75 and 0:100, w/w). Two different vegetable oils were tested as an inducer, soy bean oil and olive oil. Glycolipid biosurfatants produced from each medium formulation were extracted and quantified using high performance liquid chromatography.

Quantification of MEL's by High Performance
Liquid Chromatography (HPLC) The glycolipid quantification was carried out by subjecting the extracts to normal phase HPLC analysis on a silica gel column (i.d., packed with Zorbax Rx-SIL, 4,6 mm ID×250 mm, 5µm, Shimadzu, Kyoto, Japan) using a gradient solvent program using isocratic elution consisting of mixed acetonitrile and 2-propanol (95:5, v/v). The eluent was monitored over 20 min period at 206 nm, followed by regeneration of column for the next analysis for 10 min at a flow rate of 1 mL min −1 . The quantification of MEL was carried out by HPLC based on a standard curve using the pure MEL fraction, which was prepared by P. antarctica (Morita et al., 2009b). All measurements reported here are calculated values from at least three independent experiments.

Structure Analysis of the Purified Glycolipids, MEL's
The structures of the purified glycolipids were identified by using spectroscopy analysis, 1 H & 13 C Nuclear Magnetic Resonance (NMR) using a Varian INOVA 500 MHz at 30°C using CdCl 3 solution (Morita et al., 2011). NMR spectra were recorded under conditions as indicated on a JEOL JNM ECA-500 spectrometer.

Analysis of MEL's Glycolipid Fatty Acids
The methyl esters derivatives from the fatty acids were prepared by mixing the purified glycolipids (10 mg) with 5% v/v HCl-methanol reagent (1 mL). After the reaction was quenched with water (1 mL), the methyl ester derivatives were extracted with n-hexane and separated using gas chromatography (Shimadzu GC 2010 Plus, Cyanopropil-methyl-sil, 230°C detector temperature, 200°C injector temperature, 190°C column temperature (held for 15 min) to 230°C at 10°C min −1 , carrier gashelium. The methyl esters of the fatty acids were prepared with a modified method.

Surface Tension Measurement
The surface tension of the aqueous solution at different surfactant concentrations was measured by the Du Nouy method using an interfacial tensionmeter (Dziegielewska and Adamzak, 2013). The surface measurement was carried out at 25±1°C at the end of incubation. Each measurement was repeated 3×to give an average value.

Selection of Pseudozyma Y10BS025 as a MEL's Producer and Molecular Phylogenetic Analysis
Two yeast strains belonging to genus Pseudozyma, Y10BS016 and Y10BS025, were tested for ability to secrete MEL's. Results showed that only Pseudozyma Y10BS025 produces MEL's in significant amounts. This strain was selected as a MEL producer and was subjected to further analysis. Molecular phylogenetic analysis based on the gene encoding the D1/D2 domain of the 26S rRNA ( Fig. 1), placed the Pseudozyma Y10BS025 close to Pseudozyma hubeiensis. Nucleotide-sequence-alignment showed that the Pseudozyma Y10BS025 has 98-100% identity with other Pseudozyma strains and 100% identity with Pseudozyma hubeiensis, thus confirming its identity.

Separation of Glycolipids Produced by P. hubeiensis Y10BS025
On TLC, the P. hubeiensis Y10BS025 extract gave spots of glycolipids corresponding to MEL. The anthrone-reagent-positive spots having the same Rf values as those of the purified MEL standard (Fig. 2). The glycolipids produced by P. hubeiensis Y10BS025 showed nearly the same spots as the purified MEL standard corresponded to MEL-C and yielded higher amounts of glycolipids than the standard. The purified MEL's standard prepared from soybean oil by P. antarctica was used as a reference

Optimum Medium Formulation for MEL's Production by P. hubeiensis Y10BS025
The effects of the carbon source on MEL's production by P. hubeiensis Y10BS025 were studied using varied ratios of glucose and glycerol in the minimal basal medium and the effects of the composition of vegetable oil used as an inducer were studied by supplementing either soy bean oil or olive oil. Results showed that glycolipid biosurfactants were produced by P. hubeiensis Y10BS025 on all medium formulations tested. The optimum medium for MEL's production was the minimal basal medium with glucose: Glycerol ratio of 75:25, w/w using soybean oil as an inducer. The yield achieved was 115±3.2 g.L −1 of MEL's for 8 days of fermentation (Fig. 3). In all medium formulations tested, medium supplemented with soybean oil gave a better yield compared to that supplemented with olive oil.

Fatty-Acid Composition of MELs Produced by P. hubeiensis Y10BS025
The fatty-acid composition of the present MEL-C from soybean oil was also analyzed by the GC-MS method and compared with that of MEL-C produced by the other strains ( Table 2). The fatty-acid composition of the MEL-C produced by P. hubeiensis Y10BS025 was different from that by P. antarctica, which mainly possess two medium chain acids (C 8 to C 10 ), while those of MEL-C produced by P. shanxiensis were C 16 acids. The major fatty acids of P. hubeiensis Y10BS025 consisted of C 10 , C 14 and C 18 , with the hydrophobic structure of the present MEL-C being considerably different from those of MEL-C produced by other Pseudozyma strains. The present MEL-C produced by P. hubeiensis Y10BS025 from soy-bean oil possessed a short chain (C 2 or C 4 ) at the C-2' position and a long chain (C 8 to C 18 ) at the C-3' position of the mannose moiety. P. hubeiensis Y10BS025 was found to be a novel MEL's producer and was able to produce MEL-C with a different fatty acid composition.

Surface Active Properties of Purified MEL-C
The surface tension of MEL-C was determined by the Du Nouy method. Fig. 5 shows the surface (airwater) tension Vs concentration plot of MEL-C in distilled water. This shows MEL reduces the surface tension of water to 30.80 dyne.cm −1

DISCUSSION
Here, we show for the first time that P. hubeiensis Y10BS025 is a novel MEL-C producer. Indeed, the genus Pseudozyma has been well known to be MEL's producer (Morita et al., 2009a) and more specifically, a number of P. hubeiensis strains have also been reported Science Publications AJBB to produce glycolipid MEL's (Konishi et al., 2013). These facts indicate that the genes involved in MEL's biosynthesis should be conserved among these strains. The molecular phylogenetic tree based on the sequence of the D1/D2 domain of the 26S rRNA demonstrated that P. hubeiensis Y10BS025 is closely related to P. prolifica, Ustilago maydis and Ustilago vetiveriae. It was therefore necessary to test the ability of these microorganisms to produce glycolipids MEL's. A number of strains of Pseudozyma and Ustilago isolated from leaves and smuts of sugarcane plants have recently been identified as MEL's producers. The strains showed the ability to form abundant of MEL's using sugarcane juice as a sole nutrient source .
In this study, an optimum medium for MEL's production by P. hubeiensis Y10BS025 has been developed. This was achieved by optimizing the ratio of glucose and glycerol as carbon sources and evaluating effects of the type of vegetable oil used as an inducer. A wide range of carbon sources such as glucose, pentose, hexose, glycerol, triglycerides, fatty acids and ethanol, has been reported to support cell growth and MEL's production. Glucose (Morita et al., 2009b) and glycerol (Bhangale et al., 2013) has been used for efficient production of MEL's. To overcome the expensive cost constraints associated with MEL's production, the use of inexpensive and waste substrates for the formulation of fermentation media has been suggested (Saharan et al., 2011). Accorsini et al. (2012) used soybean oil and glycerol as low cost substrates for biosurfactant production by yeast. The use of olive oil for MEL's production has also been reported (Morita et al., 2009c). Similarly, Dziegielewska and Adamzak (2013) found that addition of 5% (v/v) of rapeseed oil increases the synthesis of MEL's from 2.20 to 12.69 g.L −1 . They also found that addition of rapeseed oil to the cultivation medium decreases the surface tension and increases both the Diameter of the Medium (DMD) and the biomass concentration.
Although from the point of view of MEL's production yield, vegetable oil has been reported as the best substrate (Morita et al., 2009b), in the present study glucose and glycerol were used as the main carbon source for MEL's production. This was intended to facilitate purification of MEL's and circumvent the need for removal of the residual oil and resulting lipase degradation products. It has been reported that when soybean oil is employed as a substrate for MEL's production, additional complicated process is required to remove the residual oil and lipase degradation products such as monoacylglycerols, diacylated glycerols and nonesterified fatty acids . To improve the efficiency of MEL production, therefore, the use of watersoluble carbon sources, such as glucose and glycerol, instead of vegetable oils is highly desirable (Morita et al., 2009c). It has been reported that the purity of glucose and glycerol-derived MEL's is higher than that of soybean oil-derived MEL's. In the extract from the culture with glucose and glycerol, byproducts such as the residual oil and its degradation products were not detected on TLC and HPLC analysis (Morita et al., 2009b). From an environmental perspective, the successful conversion of carbon glycerol to glycolipid biosurfactants by P. hubeiensis Y10BS025 in the present study has the potential to facilitate the utilization of waste glycerol.
As presented in Table 2, the fatty acid composition of MELs produced by P. hubeiensis Y10BS025 is different from those produced by P. antarctica and P. shanxiensis. This suggests that there may be different kinds of fatty acid biosynthetic pathways among MEL's producers. Differences in the steps for acetylation and fatty acid synthesis may lead to different fatty acid composition of MEL's (Morita et al., 2009b). Differences in MEL's fatty acid composition were also reported by Morita et al. (2009c). They found that the major fatty acids of the MEL-B produced from olive oil by U. scitaminea NBRC 32730 were C 8 and C 10 acids. On the other hand, the main fatty acids of MEL-B produced from olive oil by P. tsukubaensis were C 8 and C 14 . Similarly, strains of Pseudozyma producing mainly MEL-C showed different lengths of fatty acid in comparison with one another: P. hubeiensis was C 6 , C 10 , C 12 and C 16 , P. graminicola was C 6 , C 8 , C 12 and C 14 and P. shanxiensis and P. siamensis were C 2 , C 4 , C 14 and C 16 (Morita et al., 2009a). Differences in fatty acid composition shown by the present study suggest that these are novel types of MEL's. Further characterization on these glycolipids biosurfactants, therefore, may provide us with a better undestanding of the structurefunction relationships of biosurfactants.
The observed structural analyses demonstrated that the glycolipids produced from soybean oil by P. hubeiensis Y10BS025 are MEL's. However these have a different chirality on the carbohydrate moiety compared to those from P. siamensis CBS 9960 which corresponded well to the previously reported MEL-C, in which there is a different peak structure at 5.0 -5.5 ppm. The present MEL-C possesses a hydrophobic structure different from those of conventional MEL's. Accordingly, our results show that P. hubeiensis Y10BS025 has considerable potential as a MEL's producer and exhibits excellent surface activity. This study expands our knowledge on the variety of glycolipid biosurfactants and provides useful information on their structure relationships.

CONCLUSION
We conclude that the P. hubeiensis Y10BS025 is a novel MEL's producer that produces MEL-C as a majority product with mainly C-18 acids. The MEL-C produced by P. hubeiensis Y10BS025 exhibits surfacetension-lowering activity. Hence the newly isolated P. hubeiensis Y10BS025 provides high efficiency in MEL's production and would thus be highly advantageous in commercial production of promising biosurfactants.