Single cell oil of oleaginous fungi from the tropical mangrove wetlands as a potential feedstock for biodiesel
© Khot et al.; licensee BioMed Central Ltd. 2012
Received: 25 September 2011
Accepted: 2 March 2012
Published: 30 May 2012
Single cell oils (SCOs) accumulated by oleaginous fungi have emerged as a potential alternative feedstock for biodiesel production. Though fungi from mangrove ecosystem have been reported for production of several lignocellulolytic enzymes, they remain unexplored for their SCO producing ability. Thus, these oleaginous fungi from the mangrove ecosystem could be suitable candidates for production of SCOs from lignocellulosic biomass. The accumulation of lipids being species specific, strain selection is critical and therefore, it is of importance to evaluate the fungal diversity of mangrove wetlands. The whole cells of these fungi were investigated with respect to oleaginicity, cell mass, lipid content, fatty acid methyl ester profiles and physicochemical properties of transesterified SCOs in order to explore their potential for biodiesel production.
In the present study, 14 yeasts and filamentous fungi were isolated from the detritus based mangrove wetlands along the Indian west coast. Nile red staining revealed that lipid bodies were present in 5 of the 14 fungal isolates. Lipid extraction showed that these fungi were able to accumulate > 20% (w/w) of their dry cell mass (4.14 - 6.44 g L-1) as lipids with neutral lipid as the major fraction. The profile of transesterified SCOs revealed a high content of saturated and monounsaturated fatty acids i.e., palmitic (C16:0), stearic (C18:0) and oleic (C18:1) acids similar to conventional vegetable oils used for biodiesel production. The experimentally determined and predicted biodiesel properties for 3 fungal isolates correlated well with the specified standards. Isolate IBB M1, with the highest SCO yield and containing high amounts of saturated and monounsaturated fatty acid was identified as Aspergillus terreus using morphotaxonomic study and 18 S rRNA gene sequencing. Batch flask cultures with varying initial glucose concentration revealed that maximal cell biomass and lipid content were obtained at 30gL-1. The strain was able to utilize cheap renewable substrates viz., sugarcane bagasse, grape stalk, groundnut shells and cheese whey for SCO production.
Our study suggests that SCOs of oleaginous fungi from the mangrove wetlands of the Indian west coast could be used as a potential feedstock for biodiesel production with Aspergillus terreus IBB M1 as a promising candidate.
KeywordsMangrove wetlands Oleaginous fungi Single cell oil Fatty acid methyl ester Aspergillus terreus
Biodiesel as an alternative fuel has been in the forefront of the liquid biofuel sector for the last two decades. The use of edible vegetable oils such as soybean, rapeseed and non-edible oils such as Jatropha in the United States, Europe and India, respectively, as oil feedstock for biodiesel production needs to be augmented with newer technologies. To meet the demand of the biodiesel industry, alternative oil sources are being explored and developed. Recently, microbial lipids (single cell oils, SCOs) accumulated by oleaginous microorganisms e.g., microalgae and fungi, with 20% or more of their cell mass being composed of lipids, have emerged as a potential feedstock for biodiesel production [1, 2].
The applications of oleaginous fungi for biodiesel are very few although they have several advantages over conventional plant and algal resources as they can be easily grown in bioreactors, have short life cycles, display rapid growth rates, are unaffected by space, light or climatic variations, are easier to scale up and have the ability to utilize a wide range of inexpensive renewable carbon sources such as lignocellulosic biomass and agro-industrial residues. Rumbold et al (2009) have evaluated the utilization of lignocellulosic biomass hydrolysates by two yeasts (Saccharomyces cerevisiae and Pichia stipitis) and two fungi (Aspergillus niger and Trichoderma reesei) as feedstock for various industrial fermentations . Several studies reporting lipid accumulation by oleaginous yeasts and filamentous fungi on different renewable substrates such as glycerol, sewage water, whey and molasses has also been reviewed . More recently, metabolic engineering of Acinetobacter baylyi ADP1 for improved production of triacylglycerol, a source of biofuel has been studied .
Ramos et al. (2009) have shown that the biodiesel quality depends upon the fatty acid composition of the oil feedstock . For an oleaginous microbe to be considered as a suitable feedstock for biodiesel, the total lipid content (>20%) and the type of fatty acids (long chain saturated and/or monounsaturated fatty acids) are important criteria. Lipid content and fatty acid composition of SCOs varies in response to environmental factors such as type of carbon source, pH, temperature and according to the nature of microorganism i.e., it is species and strain- specific [4, 7]. This is evident from the studies on the psychrophilic oleaginous yeast Rhodotorula glacialis wherein both glucose concentration and temperature influenced the composition and degree of unsaturation of fatty acids . In Mortierella species, M. alpina was able to produce 40% (w/w) SCO while M. isabellina ATHUM 2935 gave 50.4% (w/w) oil [9, 10] when grown on glucose. The lipid production (w/w wet biomass) in n-hexadecane- and glucose-grown cells were 4.43 and 0.62%, respectively, by Aspergillus terreus MTCC 6324 . Consequently, since the accumulation of lipids by oleaginous fungi varies, not all oleaginous fungal cells can be used as a feedstock for biodiesel production. Therefore, careful selection of the oleaginous strains of the fungal species and characterization of lipid composition need to be performed to ascertain their suitability for biodiesel production.
Mangrove wetlands constitute specific regions in tropical and subtropical intertidal areas of the world where salt tolerant mangrove plants occur. Fungi from mangrove ecosystem are the second largest group amongst the marine fungi . Being a detritus based habitat, fungi from mangroves play a significant role in the decomposition processes, nutrient cycling and energy flow of the marine web. Although mangrove fungi have been reported for production of several lignocellulolytic enzymes , to the best of our knowledge, there are no reports on these marine fungi being explored for their SCO producing capability. In fact, such oleaginous fungi with their battery of lignocellulolytic enzymes could be economically relevant in the production of biodiesel from cheap renewable carbon sources. In this study, a number of fungi were isolated from three different mangrove wetland locations along the Indian west coast. The potential of the fungal cell mass of these isolates was evaluated for their lipid content and SCO profiles for biodiesel production. Fatty acid composition and some physico-chemical properties of the transesterified SCOs (biodiesel) were analyzed in order to ascertain their potential suitability as a fuel.
Results and discussion
Isolation of fungi from mangrove wetlands
Fourteen fungal isolates with different visible colony and cell morphology (as seen under the microscope) were obtained from mangrove wetlands, of which 3 were yeasts, while 11 were filamentous fungi.
Nile red staining
Oleaginous fungi accumulate high levels of lipids when carbon is in excess and a key nutrient such as nitrogen or phosphorous is limiting . These accumulated lipids or SCOs get deposited as intracellular lipid bodies (LBs) which can easily be detected by a fluorescent probe, Nile red. Kimura et al (2004) have visualized LBs using this approach, in oleaginous fungi viz., Lipomyces starkeyi IFO-10381, Rhodosporidium toruloides IFO-0559, Cryptococcus curvatus IFO-1159, Mortierella isabellina IFO7884, M.nana IFO-8794, M.ramanniana var. angulispora IFO 8187 wherein LBs varying in size, number and shape have been detected .
Biomass and total lipid (SCOs) yields
Biomass and SCO yields of oleaginous fungal isolates from mangrove wetlands of Indian west coast
Biomass (CDW ing L-1)*
Total lipid#(g g-1of CDW)*
Lipid yield#( g L-1)
4.27 ± 0.1a
0.11 ± 0.02ab
0.25 ± 0.01a
0.47 ± 0.02a
1.07 ± 0.1a
4.14 ± 0.3a
0.15 ± 0.02ab
0.28 ± 0.01a
0.62 ± 0.01a
1.16 ± 0.3ab
5.84 ± 0.5bc
0.17 ± 0.04b
0.30 ± 0.05a
0.99 ± 0.02b
1.75 ± 0.2bc
6.44 ± 0.3c
0.09 ± 0.01a
0.30 ± 0.03a
0.57 ± 0.02a
1.93 ± 0.2c
5.52 ± 0.2b
0.10 ± 0.01a
0.54 ± 0.03b
0.552 ± 0.3a
2.98 ± 0.3d
Biomass and SCO yields of the other oleaginous filamentous fungi studied for biodiesel production have been found to be 4.17 g L-1 in 96 h and 13.6 g L-1 in 48 h with a lipid productivity of 23% and 23.3% for Mucor circinelloides MU241, and Aspergillus sp. respectively, while a SCO yield of 53% (w/w) of dry cell mass was obtained for Mortierella isabellina, when grown on glucose with concentrations ranging from 2% to 7% [7, 18, 19]. Aspergillus terreus MTCC 6324 exhibited 4.43% of lipid productivity when grown on n- hexadecane .
Fatty acid profiles of fungal SCOs
Fatty acid methyl ester composition of the SCOs of oleaginous mangrove fungal isolates
FAME composition (% of total fatty acid)*
Biodiesel properties of transesterified fungal SCOs (FAMEs)
Biodiesel properties of the transesterified SCOs of oleaginous fungi from mangrove wetlands
US biodiesel standard ASTM D6751
EU biodiesel standard EN 14214
Indian biodiesel standard IS 15607
Density * (g.cm-3)
0.86 – 0.90
0.86 – 0.90
Kinematic viscosity# (40 °C; mm2s-1)
1.9 – 6.0
3.5 – 5.0
3.5 – 5.0
HHV (MJ kg -1)**
TAN (mg KOH/g) *
Concentration of linolenic acid (C18:3) (%) *
FAME having ≥4 double bonds (%)*
Iodine value (IV), saponification number (SN) and higher heating value (HHV) are three important chemical properties of biodiesel attributed to the fatty acid profile. The IV is a crude measure of degree of unsaturation of the biodiesel and is often used in connection with its oxidative stability. The SN indicates the amount of TAG present in total lipid and HHV depends upon both IV and SN. Therefore, in the present study the SN and IV were experimentally determined as well as calculated empirically from fatty ester composition of transesterified total lipids. The experimentally determined and the predicted IVs were below the EN14214 specification (120 max) and suggest good oxidative stability of the transesterified oils from mangrove fungi. The calculated and experimentally determined SNs were found to be comparable for all the isolates while HHVs of ~ 40 MJ kg-1 were similar to methyl esters of vegetable oils .
For biodiesel, CN has been found to increase with an increasing weight percentage of saturated and long chain fatty ester. In fact, methyl esters of stearic acid (C18:0), which is of relevance to biodiesel, have been found to possess the highest CN (> 80) [31, 32]. In the present study, methyl esters of long chain saturated fatty acids namely stearic acid (C18:0) and palmitic acid (C16:0) were found to be the major components in all transesterified fungal oils. The calculated CNs for all the mangrove isolates were found to be between 56-61(Table 3), and comparable with the predicted values reported for biodiesel obtained from other oleaginous fungi . These values are in the acceptable range of international biodiesel standard norms suggesting their possible suitability as a fuel.
The total acid number (TAN) as determined according to EN14214 was estimated to be 0.1-0.2 mg KOH g-1 for isolates IBB G4, G5 and M1 while it was negligible in IBB B2 and F14 and were in accordance with the biodiesel standards. These TAN values were much lower as compared to biodiesel from vegetable oils which lie between 0.08-0.62 mg KOH g-1 and algal oils, (0.37-0.71 mg KOH g-1) . Free fatty acid (FFA) content determined as per EN 14214 was found to be < 0.1% for all 5 isolates. Other chemical properties of biodiesel evaluated were the concentration of linolenic acid (C18:3) and wt % of FAMEs having ≥ 4 double bonds. From the fatty acid profiles of fungal SCOs, it can be seen that the concentration of C18:3 were well below the specified limit of 12 max and fatty esters with ≥ 4 double bonds were not detected in transesterified oils of mangrove fungi (Tables 2 and 3).
Effect of different glucose concentrations and cheap renewable substrates on SCO yields of IBB M1
Growth and lipid (SCO) yields by strain IBB M1 at differing initial glucose concentrations
Glucose (g L-1)
Biomass (CDW in g L-1)*
Lipid yield (g L-1)
Lipid content (g g-1of CDW )*
Residual glucose (g L-1)
Lipid/glucose yield coefficient (g g-1)
4.6 ± 0.3 a
2.28 ± 0.09 a
0.49 ± 0.03 a
0.16 ± 0.02a
0.23 ± 0.01 a
5.5 ± 0.1 b
3.00 ± 0.13 b
0.54 ± 0.02 a
0.18 ±0.01 ab
0.10 ± 0.03 b
5.3 ± 0.2 bc
2.60 ± 0.1 a
0.49 ± 0.02 a
0.18 ±0.01 ab
0.05 ± 0.01c
4.9 ± 0.1 ac
2.35 ± 0.08 a
0.48 ± 0.05 a
0.19 ± 0.009 b
0.03 ± 0.009 c
4.0 ± 0.3 d
1.93 ± 0.2 c
0.48 ± 0.04 a
0.25 ±0.014 c
0.02 ± 0.01 c
The ability of strain IBB M1 to accumulate lipids on locally available cheap carbon sources was also evaluated. The agro-residues, sugarcane bagasse, grape stalks and groundnut shell are the major lignocellulosic materials in tropical countries such as India and are readily available. Cheese whey as another commonly available cheap renewable substrate was also tested. Preliminary studies showed that the lipid yields of IBB M1 when cultivated using milled grape stalk, groundnut shell, sugarcane bagasse (1%, w/v) and cheese whey were 3.62, 10.56, 16.63 g 100 g-1 DW of substrate and 13.1 g 100 g-1 CDW respectively after 72 h. Thus IBB M1 was able to accumulate SCO to varying degrees on these cheap renewable substrates. To date, to the best of our knowledge, no known reports on milled sugarcane bagasse, grape stalk and groundnut shell for SCO production by filamentous fungi exist. Subramaniam et al (2010) have reviewed other low cost feedstocks for microbial lipid production which include industrial glycerol, corn steep liquor, molasses, sweet sorghum, municipal wastewater and effluents . Recently an oleaginous yeast Yarrowia lipolytica has been shown to produce SCO (58.5%) on sugarcane bagasse hydrolysate medium  while Zygomycetous fungi have been shown to produce significant quantities of biomass and SCO when grown on cheese whey . Thus, the lipid yields obtained on the cheap substrates evaluated in the present study are of importance as preliminary data indicates that IBB M1 was able to utilize these lignocellulosic wastes for SCO production.
Identification of fungal isolate IBB M1
An earlier report by Singh and Sood  suggested that Aspergillus terreus Thom 309 had high SCO producing ability, but to date has been not explored for its biodiesel potential. A total lipid content of 51% (w/w) has been reported for A. terreus Thom 309 similar to that of IBB M1 but with a different FAME profile. A. terreus IBB M1 exhibited suitable fatty acid profiles for biodiesel with high stearic acid content (23.6%) and low linolenic acid content (0.4%) while the earlier report (A. terreus Thom 309) showed 0.3% and 20.7%, respectively. Higher contents of long chain monounsaturated fatty acid esters and negligible amounts of PUFAs are desirable for fuel properties such as CN and IV specified in biodiesel standards and these criteria seem to be fulfilled by IBB M1.
In the present study, the SCOs of fungal cell mass from the mangrove wetlands of Indian west coast were explored as biodiesel feedstock. Five isolates of oleaginous filamentous fungi were obtained with the neutral lipid as the major component of their total lipids. The presence of higher quantities of saturated and monounsaturated C16 and C18 fatty acids and the absence of long chain PUFAs were the major features of the SCOs of these tropical mangrove fungi. The experimentally determined and predicted biodiesel properties based on FAME composition of the fungal SCOs of 3 isolates are found to lie within the range specified by international biodiesel standard specifications. Aspergillus terreus IBB M1 with the highest yield of SCO was identified as a promising strain for further studies. Preliminary studies suggest that Aspergillus terreus IBB M1 from the mangrove wetlands is able to utilize cheap renewable substrates such as cheese whey and agro-residues viz., sugarcane bagasse, grape stalk and groundnut shell for SCO production. It is currently being studied further with respect to utilization of agricultural residues in order to optimize the productivity of cell mass rich in SCO. Thus, oleaginous fungi from the mangrove ecosystem with their varied lignocellulolytic enzymes could play a key role in the overall economics of biodiesel production.
Analytical grade solvents and chemicals viz., chloroform, methanol, acetone, formaldehyde, KH2PO4, Na2HPO4.12H2O, anhydrous sodium sulphate, NaCl, KCl, and MgCl2 were purchased from Merck Ltd., Mumbai, India. Czapek-Dox medium was obtained from HiMedia laboratories, Mumbai, India while 1, 1, 1-trichloroethane, from National Chemicals, Vadodara, India. Silicic acid, chromatography grade and Nile red were purchased from Sigma-Aldrich, Inc., USA. ‘Bead beater’ was procured from Biospec Products, Inc., USA. TAN and FFA content were determined using Kittiwake DIGI Biodiesel test kit, purchased from Kittiwake Developments Ltd., UK. Iodine solution (Wijs) was purchased from Acros Organics, Belgium while alcoholic KOH solution was procured from Merck, Germany. Potassium iodide and phenolphthalein were obtained from Fisher Scientific, Mumbai, India. All other chemicals obtained locally were of AR grade and at least 98% pure according to the manufacturer.
Sampling sites and isolation of mangrove fungi
The present study was carried out on 14 fungal strains which were isolated from mangrove wetlands of the Indian west coast located at Bandra in Mumbai (19°3′N latitude and 72°49′E longitude), Murud (18°18′N latitude and 72°58′E longitude) and Mandovi estuary in Goa (15°29′N latitude and 73°50′E longitude). The sampling was done in December 2007 and May 2008. The soil samples rich in detritus material were collected from the base of the mangrove trees in sterile 50 mL screw-cap tubes. The soil samples (1 g) were weighed and inoculated into 50 mL of Czapek Dox broth and incubated with shaking (180 rpm) for 72 h at 28°C. After incubation, this broth was used to streak Czapek Dox agar plates to obtain the isolates. Penicillin and streptomycin were added to the media at concentrations of 400 U mL-1 and 1mg L-1, respectively, to prevent bacterial growth.
Media and incubation conditions
Czapek Dox basal medium used for isolation contained (gL-1 ), 30.0 sucrose, 3.0 NaNO3, 1.0 K2HPO4, 0.5 MgSO4.7H2O, 0.5 KCl and 0.01 of ferrous sulphate supplemented with 15 g L-1 of NaCl, 6.7 mg L-1 of ZnSO4.7H2O and 1 mg L-1 Co[NO32.6H2O. Isolated fungal strains were maintained on the above Czapek Dox agar slants at 4°C. The lipid fermentation medium used during all screening stages of the mangrove fungal isolates was carbon rich and nitrogen limited to induce lipid accumulation and contained (g L-1), 30.0 glucose, 1.5 yeast extract, 15.0 NaCl, 0.5 NH4Cl, 5.0 Na2HPO4.12H2O, 7.0 KH2PO4, 1.5 MgSO4.7H2O, 0.1 CaCl2.2H2O, 0.01 ZnSO4.7H2O, 0.08 FeCl3.6H2O, 0.1 CuSO4.5H2O, and in mg L-1, 0.1 Co[NO32.6H2O, 0.1 MnSO4.5H2O and pH adjusted to 5.5 . The abovementioned media was inoculated with 1 x106-108spores mL -1 and incubated on a rotary shaker (120 rpm) at 30°C for 72 h. The fungal cell mass was harvested by filtration and washed thrice with distilled water.
Batch experiments were performed in conical flasks in the abovementioned medium containing initial glucose concentrations ranging from 10–100 g L-1 as well as on locally available cheap waste substrates viz., cheese whey and agro-residues including sugarcane bagasse, ground nut shell and grape stalks. The agro-residues were washed, dried, milled to pass through a 1 mm sieve and added to the medium as a sole carbon and energy source (1% w/v). All other cultivation and incubation conditions were the same as mentioned above.
Nile red staining
All the fungal strains were checked for intracellular LBs indicative of lipid accumulation by Nile red fluorescence staining[15, 40]. Microscopy was performed with a Nikon Eclipse 80i light microscope, equipped with a digital camera using a 465–495 nm excitation filter, a 505 nm diachronic mirror and a 515–535 nm barrier filter.
Extraction and gravimetric determination of total lipids as SCO
The harvested and washed cell mass was freeze dried. The dried biomass was crushed to powder, the dry weight (g) measured gravimetrically and used for lipid extraction which was performed according to Schneiter and Daum (2006) after cell disruption . Briefly, two techniques were used for disruption of fungal cells in order to determine optimal cell lysis for extraction of intracellular lipids. The first technique employed a ‘bead beater’ and the second one used liquid nitrogen. For bead beater extraction, a known dry weight of fungal biomass was transferred to a 20 mL chamber of the bead-beater filled up to its 3/4th volume with 0.5 mm acid washed glass beads. Methanol (10 mL) was added to fill the rest of the chamber and 9–20 bead beating cycles (each of 1 min) were carried out in cold conditions till cell breakage was confirmed by microscopy. For disruption by liquid nitrogen, dry biomass sample was transferred to a chilled mortar pestle containing glass beads to which 10 mL methanol was added and the sample finely ground. In both the techniques, the homogenate obtained after cell disruption was collected, transferred to glass stoppered flask and processed . The final extracted organic phase obtained was transferred to a pre-weighed round bottom flask and evaporated in a rotary evaporator at 55°C under vacuum. The flask containing total lipid extract was weighed and the total lipid extract as SCO was gravimetrically determined. The total lipid extract was reconstituted in 5–6 mL chloroform / methanol (2:1, v/v) and stored in clear screw top glass vials at −20°C till further use.
Fractionation of SCOs
The total neutral lipid content in the extracted SCOs of five oleaginous fungal isolates was determined by lipid fractionation . Briefly, a known weight of total lipid extract was dissolved in chloroform (1 mL) and fractionated using a column (25 mm x 100 mm) of silicic acid (1 g), activated by heating overnight at 110°C. Successive applications of 1,1,1- trichloroethane (100 mL) , acetone (100 mL) and methanol (50 mL) were carried out for elution of neutral lipids, glycolipids plus sphingolipids and phospholipids, respectively. The weight of each fraction was determined after evaporation of the respective solvent.
Preparation and analysis of FAMEs from fungal SCOs
To analyze the fatty acid profile of the SCOs of oleaginous tropical mangrove fungi, the transesterification was carried out according to Leung et al. (2010) . The reaction was carried out in a 50 mL round bottom flask kept in a thermostatic bath with a reflux condenser and a magnetic stirrer using a methanol to oil molar ratio of 60:1 and a catalyst (NaOH) concentration of 1.5-3 wt % relative to SCO. The reaction was carried out under ambient pressure for 90 minutes at 60°C. The mixture was allowed to stand for 1 h to collect the upper organic layer (FAMEs) and the solvent was removed by rotary evaporator (Büchi, Rotavapor RE120, 70°C). The FAME samples were reconstituted in chloroform/ methanol (2:1, v/v) and stored in clear screw top glass vials at −20°C till further use.
To obtain the fatty acid profile of the transesterified fungal SCOs, all the samples were appropriately diluted with chloroform/ methanol (2:1, v/v) mixture and the sample analyzed using gas chromatograph as per the AOAC standard method . A capillary column CP-Sil88 of 0.25 mm ID and 50 m length with flame ionization detector (GC1000, Chemito) was used. The column temperature was programmed as being upgraded from 100°C to 198°C at a rate of 1.5°C /min and held for 8 min. Nitrogen was used as the carrier gas. The injector and detector temperatures were set at 225°C and 250°C, respectively. Identification of peaks was performed by comparison with authentic FAME standard mixture and quantifications done on the basis of their specific peak areas.
Determination of biodiesel properties of SCOs
Different physicochemical fuel properties namely density, kinematic viscosity, SN, IV, HHV, CN, TAN and FFA were determined experimentally as well as by using predictive models and mathematical equations for the transesterified SCOs of the five oleaginous fungal isolates.
where ci is the concentration (mass fraction) and ρi is the density of component (individual fatty acid methyl ester) present in biodiesel.
where ν mix = the kinematic viscosity of the biodiesel sample (mixture of fatty acid alkyl esters), Ac = the relative amount (%/100) of the individual neat ester in the mixture (as determined by, GC-FID) and vc = obtained from the kinematic viscosity database of individual FAME present in biodiesel .
where CNi represent reported CN of pure fatty acid methyl ester available in database and Wi is the mass fraction of individual fatty ester component detected and quantified by GC-FID.
Identification of fungal isolate IBB M1
The filamentous fungal isolate IBB M1 was identified in the laboratory by both morphological and molecular approaches. Identification was based on macroscopic observation of the colonies and examination of the microstructural characteristics using universal identification keys for fungi . Scanning electron microscopy analysis was carried out to study the microscopic characteristics (Stereoscan 440, LEO/Leica, Cambridge, UK).
In molecular techniques for identification, genomic DNA of the fungal strain IBB M1 was isolated by using the standard protocol . Partial region of SSU rDNA was amplified by PCR using universal fungal primers, NS1 (5’-GTAGTCATATGCTTGTCTC-3’) and NS4 (5’-CTTCCGTCAATTCCTTTAAG-3’) of 1,100 bp . PCR products were purified by using gel extraction kit (GeNei, Bangalore, India) and sequenced using the Big Dye Terminator cycle sequencing kit (Applied Biosystems, USA) according to the manufacturer’s protocol followed by purification using Big Dye X-Terminator Purification kit (Applied Biosystems, USA) and analyzed in a DNA Analyzer (3730 DNA Analyzer, Applied Biosystems, USA). Sequence data were analysed using Sequence analysis software. The 18 S sequence obtained was aligned using BLAST algorithm to find matches within the non redundant database at NCBI (National Centre for Biotechnology Information; http://blast.ncbi.nlm.nih.gov/Blast.cgi) . Sequence data were submitted to GenBank through submission tool BankIt of NCBI.
All values are means of three independent experiments. Statistical analyses were performed using SPSS 11.5 statistics software (SPSS Inc., Chicago, IL, USA). Means were compared and analyzed using either t-test or one-way analysis of variance (ANOVA) with Tukey HSD post hoc multiple comparison test. Differences were considered statistically significant for p < 0.05.
Cell Dry Weight
Fatty Acid Methyl Ester
Free Fatty Acid
Gas Chromatography Flame Ionization Detector
Higher Heating Value
Polyunsaturated Fatty Acid
Single Cell Oil
Total Acid Number.
The authors thank the Institute of Bioinformatics and Biotechnology (IBB), University of Pune, Pune for the financial support provided to carry out this work. MK and SK were funded by IBB and Department of Biotechnology, New Delhi, respectively.
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