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Biodiesel production from isolated oleaginous fungi Aspergillus sp. using corncob waste liquor as a substrate
Bioresource Technology 102 (2011) 9286–9290
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Biodiesel production from isolated oleaginous fungi Aspergillus sp. using corncob waste liquor as a substrate G. Venkata Subhash, S. Venkata Mohan ⇑ Bioengineering and Environmental Centre (BEEC), Indian Institute of Chemical Technology (IICT), Hyderabad 500 607, India
a r t i c l e
i n f o
Article history: Received 27 April 2011 Received in revised form 22 June 2011 Accepted 23 June 2011 Available online 30 June 2011 Keywords: Transesterification Fatty acid methyl ester (FAME) Saturated fatty acid (SFA) Substrate degradation
a b s t r a c t The study documented the potential of isolated filamentous fungus Aspergillus sp. as whole cell biocatalyst for biodiesel production using Sabourauds dextrose broth medium (SDBM) and corncob waste liquor (CWL) as substrates. SDBM showed improvement in both biomass production (13.6 g dry weight/ 1000 ml) and lipid productivity (23.3%) with time. Lipid extraction was performed by direct (DTE) and indirect (IDTE) transesterification methods. DTE showed higher transesterification efficiency with broad spectrum of fatty acids profile over IDTE. CWL as substrate showed good lipid productivity (22.1%; 2 g dry biomass; 48 h) along with efficient substrate degradation. Lipids derived from both substrates depicted high fraction of saturated fatty acids than unsaturated ones. Physical characteristics of fungal based biodiesel correlated well with prescribed standards. CWL derived biodiesel showed relatively good fuel properties (acid number, 0.40 mg KOH/g of acid; iodine value, 11 g I2/100 g oil; density, 0.8342 g/cm3) than SDBM derived biodiesel. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Some of the microorganisms such as bacteria, microalgae, fungi, etc. are reported to be oleaginous which can accumulate significant amounts of lipids (characteristically similar to that of vegetable oils) with good fraction of saturated fatty acids (SFA) than unsaturated fatty acids (USFA) (Papanikolaou et al., 2004; Wahlen et al., 2011; Venkata Mohan et al., 2011). Microorganisms metabolically transform the external carbon into carbohydrate or hydrocarbon and then to lipids. Lipids are considered to be important storage compounds in the form of triacylglycerols (TAG) and esters. If the lipid content in the cell exceeds 20%, then microorganism can be called as oleaginous microorganism. Actinomycetes group under restricted growth conditions show higher lipid productivity (70%) and accumulate them intracellularly as TAGs (Chisti, 2007). Few bacteria are reported to accumulate complex lipid in the form of polyhydroxyalkanoates (Steinbu and Fu, 1998). Yeast strains such as Rhodosporidium sp., Rhodotorula sp., and Lipomyces sp. showed intracellular lipids accumulation as high as 70% of their biomass dry weight (Kavadia et al., 2001). Substrate feeding modes show marked influence on the lipid productivity of oleaginous yeast Rhodosporidium toruloides Y4 (Zhao et al., 2011). In fungi, the storage lipids usually contain TAG with good percentage of SFA during stationary phase within special organelles known as lipid granules (Aggelis et al., 1995). Lipid accumulation abilities ⇑ Corresponding author. Tel.: +91 40 27191664. E-mail address: [email protected] (S. Venkata Mohan). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.06.084
of two endophytic fungal isolates Colletotrichum sp. and Alternaria sp. grown under optimum and nutrient-stress conditions were studied by Dey et al. (2011). Lipid content varies according to the nature of microorganism (biocatalyst), culture conditions (such as temperature, pH, nutrients, culture time, etc.) and substrate (Alvarez and Steinbuchel, 2002; Papanikolaou et al., 2004). Lipid profiles could also be modified by genetic engineering of oleaginous microorganisms (Vicente et al., 2010). The major advantage of microbial derived lipids are their ability to use inexpensive organic materials as the substrate, no need for arable land for their growth, does not lead to the food vs fuel crisis and their inherent feasibility of upscaling with practical viability. In this communication we have evaluated the potential of isolated fungus Aspergillus sp. as whole cell biocatalyst/feedstock for the production of lipids using corncob waste liquor (CWL) as primary substrate. An attempt was also made to study the changes in composition of fatty acid methyl ester (FAME) after transesterification of fungal oil by indirect and direct methods. 2. Experimental methodology 2.1. Fungal biocatalyst Fungal strain was isolated from the marshy soil in our institute. One gram of soil was aseptically transferred to 10 ml of sterile distilled water in laminar air chamber. Dilutions of 10 4 and 10 5 were spread on potato dextrose agar medium (PDA; pH 5.5; Hi-media) and incubated (28 ± 2 °C) for 36 h. After incubation four
G. Venkata Subhash, S. Venkata Mohan / Bioresource Technology 102 (2011) 9286–9290
fungal colonies were observed. Among them a single colony was isolated based on the morphological characteristics viz., color, texture and mycelium formation. The fungal isolate was stained (colony and spores) with Lactophenol Cotton Blue (Murray et al., 1995) and visualized as smears with epifluorescent microscope (Nikon Eclipse-80i) at 400 magnification (Supplementary Fig. 2). The images were captured on encompassed digital camera (YIM-smt, 5.5 mega pixels) using NIS-elements (D3.0) software. Lactophenol Cotton Blue and microscopic observations also confirmed the isolate belongs to Aspergillus sp. Further, this isolate was used as whole cell biocatalyst for lipid extraction. The pure fungal strain was sub-cultured onto PDA slants and incubated for growth (36 h) at 28 ± 2 °C prior to their usage in the experiments. 2.2. Corncob waste liquor (CWL) CWL that was obtained after acid (2% HCl, pH, 5) and thermal (121 °C for 45 min at 15 lbs pressure) treatment of corncob (COD, 5000 mg/l; VFA, 536 mg/l; TSS, 150 mg/l; reducing sugars, 882 mg/l; carbohydrates, 567.24 mg/l and color, 400 hazen units) was used as substrate for harnessing biodiesel. 2.3. Fungal Lipid production The detailed experimental methodology designed and executed as depicted in Supplementary Fig. 1. Aspergillus sp. was inoculated into 500 ml flasks containing 250 ml of sterile Sabourauds dextrose broth medium [SDBM; 40 g glucose, 10 g peptone in 1000 ml of water (pH 5.0)] and kept in a rotary shaker incubator (150 rpm) at 30 °C. Initially biomass and lipid productivity was optimized using SDBM culture. At regular 12 h time intervals (12, 24, 36, 48, 60, 72, 84 and 96 h) fungal biomass was collected by centrifuging the mycelium at 10000 rpm for 15 min and biomass growth (wet weight (g)) was estimated gravimetrically. Following biomass concentration, the cell precipitate was washed with distilled water (three times) and allowed to dry in oven at 60 °C for sufficient time. After drying, the dried biomass was crushed to powder using piston-motor and dry weight (g) was measured. The resulting dried fungal biomass acquired from different time intervals was used for lipid extraction. Two different types of transesterification procedures viz., single and two-step were employed and their efficiencies of extraction and conversion were evaluated. Based on the initial optimization experiments using SDBM, lipid production was further studied with the same isolate using CWL as substrate (5000 mg COD/l) and kept for growth under same optimum conditions obtained during preliminary/primary study (pH, 5; 30 °C, 150 rpm, 48 h incubation). The dried fungal biomass was subjected to lipid extraction. 2.3.1. Transesterification Two types of transesterification procedures were employed for the conversion of fungal lipids to fatty acid methyl esters (FAME) viz., direct transesterification of biomass (i.e. without the initial extraction step (single-step transesterification) and indirect transesterification by initial extraction of lipids from biomass followed by transesterification (two-step transesterification) (Vicente et al., 2010; Lewis et al., 2000). 2.3.1.1. Direct transesterification (DTE). Transesterification reaction mixture (methanol:hydrochloric acid:chloroform (10:1:1 (v/v/v); 12 ml) was added to dried fungal biomass (200 mg) in screw-cap test tube and subjected to sonication (5 min; 20 kHz) to facilitate cell membrane disruption (Lewis et al., 2000). Presonicated cells were suspended in the solution, vortexed and immediately placed at 90 °C under stirring (300 rpm) for 8 h in water bath. At the end of reaction, the mixture was diluted with distilled water (DW) and
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then extracted with ethyl acetate (EA; 2 100 ml). The EA layer was washed with DW to bring the solution pH to neutral. The two immiscible layers of DW and EA were separated using separating funnel and the solvent mixture was dried over anhydrous sodium sulfate and evaporated in a rotary evaporator. The residual FAME’s were weighed to calculate the yield and then analyzed to determine its composition. 2.3.1.2. Indirect (two-step) transesterification (IDTE). Dried fungal biomass (200 mg) in screw-cap tube was subjected to addition of each solvent in sequence (for 30 s) viz., chloroform, methanol, water ((1:2:0.8 (v/v/v)) and the resulting mixture was subjected to sonication (5 min; 20 kHz) for disruption of fungal cell wall. Biomass-solvent mixture after 18 h (with occasional shaking) was phase separated using a glass funnel. Total lipid extract (recovered from upper chloroform phase) was used for FAME preparation. Acid transesterification was done by adding the mixture of concentrated sulfuric acid–methanol (1 ml:100 ml/l of oil) to the preheated fungal oil and kept for stirring (200 rpm; 35 °C) for 1 h followed by 24 h incubation at room temperature (200 rpm). After incubation, the reaction mixture was allowed to stand for 1 h to separate the top layer with excess sulfuric acid (recover impurities) and the lower phase with oil. The lower oil layer is further subjected to alkali transesterification with sodium methoxide solution [sodium hydroxide (6.5 g/l of oil) in methanol (150 ml/l of oil)]. During reaction startup, half of the reaction mixture was added to the oil and kept for heating (55 °C; 200 rpm) for 1 h followed by cooling (room temperature; 24 h). After transesterification, the solvent was partially removed and extracted with EA (2 100 ml). After separating the top (mixture of FAME dissolved in EA) layer, the mixture was washed with DW for neutralization and the solvent was dried over anhydrous sodium sulfate followed by rotary evaporation. 2.4. Analysis The broth and CWL were evaluated through chemical oxygen demand (COD), pH and ORP analysis according to the procedures outlined in standard methods (APHA, 1998). The composition of concentrated FAME samples were detected by gas chromatography (GC; Shimadzu 17A) using flame ionization detector (FID), capillary column [SE 30 (30 0.32 mm)] and nitrogen as carrier gas (flow rate, 1 mL/min) [oven temperature; 140 °C (initially for 5 min), increased to 240 °C (ramp of 4 °C/10 min); injector temperature: 280 °C; detector temperatures 300 °C; split ratio: 1:10]. FAME composition was identified by comparing the retention time (RT) with authentic standards (FAME mix; SUPELCO; LB66766) and quality was compared with ASTM standards (ASTM, 2002). The quality of the fungal biodiesel was determined by analyzing iodine value (IV) (D 1959-97), acid number (D664) and density (ASTM; D6751-02). 3. Results and discussion 3.1. Biomass and lipid productivity with SDBM Fungal isolate used as whole cell biocatalyst showed good biomass growth with concomitant lipid accumulation by consuming available carbon and other nutritive compounds present in SDBM (Supplementary Fig. 3). Lipid accumulation showed a gradual increment up to 48 h followed with a drop which continued up to 96 h (Fig. 1). Fungal growth was relatively slow in between 0 and 12 h which also resulted in lower lipid accumulation. The wet/dry (W/D) biomass at this particular phase was 24/6 g with a lipid productivity of 10.2% (based on dry weight). A good
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Table 1). Both the methods showed higher fraction of SFA than USFA which is considered as a potential feature to indicate the fuel quality of fungal based diesel. DTE method evidenced good fatty acid profile comprising of 10 SFA and seven USFA in agreement to its high lipid productivity while IDTE owing to its low lipid productivity showed a moderate fatty acid profile depicting six SFA and three USFA. Presence of higher SFA indicates the conversion efficiency of the transesterification method to convert fatty acids into methyl esters. Though the amounts of fatty acids are less in IDTE method, the existence of less number of USFA indicates its negligible spin towards unsaturation. Marginal variation was observed between the SFA profile of both methods. DTE method additionally showed the presence of lauric acid methyl ester (C12:0), tridecanoic acid methyl ester (C13:0), palmitic acid methyl ester (C16:0) and behenic acid methyl ester (C22:0). Presence of additional SFA indicates the efficacy of DTE method than IDTE towards acquiring high fuel and lubricating properties. The higher yield of lipid profile with DTE method might be attributed to the improved amount of total lipids extracted from submerged culture (Lewis et al., 2000). This observation is supported by previous report depicting increased recovery of fatty acids from microorganisms by DTE method (Dionisi et al., 1999). Solvent addition strategy in sequence significantly influences the extraction efficiency of lipids. Adding solvents in the order chloroform–methanol–water showed better extraction (Smedes and Askland, 1999).
Fig. 1. Lipid profile in relation to dry biomass weight with the function of time.
increment in both biomass growth (W/D; 48/7.80 g, 60/8.8 g) and lipid productivity (12.42%, 18.34%) was obtained at 24 and 36 h, respectively. Maximum biomass growth (72/13.6 g (W/D)) and lipid productivity (23.33%) was registered at 48 h. Further increment in cycle period showed a gradual decrement in the lipid accumulation capability of fungal isolate [17.42% (60 h), 15.45% (72 h), 12.32% (96 h)] due to utilization of reserved food granules (including lipids) in absence of available carbon source. Growth of fungus attained stationary phase at 48 h. Under carbon deprived conditions over a prolonged period, the fungal hyphae undergoing a stationary growth phase which establishes balance between hyphal mass tending to either increment or decrement. Increment in lipid productivity was found to depend on combined influence of biomass growth and substrate availability.
3.2. Lipid productivity with CWL Alternatively, CWL was also evaluated as substrate (main carbon source) for fungal-lipid production. This approach facilitates utilization of waste for value-added product recovery with simultaneous treatment. In this case the optimum conditions obtained during primary study performed with SDBM were employed. Fungi showed effective biomass growth as well as lipid production with CWL. Lipid productivity of 22.1% (DTE method per dry biomass) was observed along with 15/2 g (W/D) of biomass growth at 48 h. Similar to SDBM, CWL based lipids also documented higher fraction of SFA compared to USFA (Table 1; Supplementary Table 1). On the contrary, CWL showed relatively good fatty acid profile compared to SDBM. SFA profile showed the presence of additional fatty acid (caprylic acid methyl ester (C8:0)) with CWL-lipid
3.1.1. Transesterification influence on FAME profile All the available microbial lipids are not suitable for production of biodiesel. Only lipids with fatty acid ester linkages (also referred to as saponifiable lipids) and free fatty acids can produce FAME, which can be used as biodiesel. During transesterification with methanol, both microbial lipids and free fatty acids can be converted to FAME in presence of a suitable acid catalyst. Indirect and direct transesterification methods were evaluated for the preparation of fungal based FAME. The proportions of SFA and USFA extracted from the fungal-lipid varied with the function of applied transesterification methods (Table 1; Supplementary
Table 1 Characterization of fungal lipid extracted from SDBM and CWL. Physical properties of biodiesel
Biodiesel standards*
<0.5 Acid number (mg KOH/g of acid) Iodine value <25 (g of I2/ 100 g of oil) Density (g/ 0.8800 cm3) Type of SFA (%, in relation to the total fatty acid) Type of USFA (%, in relation to the total fatty acid) *
ASTM (2002).
SDBM-lipid
CWL-lipid
Direct transesterification method (DTE)
Indirect transesterification method (IDTE)
Direct transesterification method (DTE)
0.42
0.47
0.40
16.0
12.0
11
0.8012
0.8241
0.8342
C10:0 (0.31), C12:0 (0.37), C13:0 (0.04), C14:0 (0.19), C15:0 (0.10), C16:0 (0.73), C17:0 (29.6), C18:0 (48.7), C20:0 (2.99), C22:0 (0.06)
C8:0 (2.96), C10:0 (2.4), C14:0 (0.70), C15:0 (0.28), C18:0 (56.6), C20:0 (1.12)
C8:0 (0.17), C10:0 (0.31), C12:0 (0.36), C13:0 (0.04), C14:0 (0.19), C15:0 (0.2), C16:0 (0.73), C17:0 (22.45), C18:0 (43.4), C20:0 (12.2), C22:0 (2.9)
C14:1n9c (0.1), C16:1n9c (14.4), C18:1n9t (1.60), C18:1n9c (0.11), C18:2n6c (0.42), C18:3n3 (0.09), C22:1n9 (0.089)
C16:1n9c (16.8), C18:2n6c (4.5), C22:1n9 (14.42)
C16:1n9c (14.8) C18:1n9t (0.42) C18:1n9c (0.6) C18:2n6c (0.42)
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compared to SDBM-lipid. While the USFA profile with CWL showed the presence of myristoleic acid methyl ester (C14:1n9c), linolenic acid methyl ester (C18:3n3) and erucic acid methyl ester (C22:1n9)). However, SDBM-lipid documented the presence of additional three USFA (myristoleic acid methyl ester (C14:1n9c), Cis-11-eicosenoic acid methyl ester (C20:1), erucic acid methyl ester (C22:1n9)). All the SFA and USFA detected with CWL-lipid were noticed to possess good lubrication and less unsaturation properties. Apart from the mentioned features the fatty acids derived also have potential and are used in making of pharmaceuticals and lubricating agents. Lipid productivity and fungal biomass growth obtained from this study documented the potential of Aspergillus sp. as whole cell biocatalyst to produce good quality biofuel utilizing CWL as substrate. Fig. 2. Substrate (COD) degradation profile during lipid production.
3.3. Fuel characterization of fungus lipids The lipids extracted from fungus by two transesterification methods evidenced good fuel characteristics but varied with the methods employed (Table 1). Acid number (AN), iodine value (IV) and density were in accordance with the biodiesel standards indicating the potential of fungal oil to use as biodiesel. DTE showed efficient fuel properties compared to IDTE. DTE method showed low AN (0.42 mg KOH/g of acid) compared to IDTE (0.47 mg KOH/g of acid). Low AN value indicates non-corrosive nature of the fuel. Similarly, IV of fungal oil was found to be low with DTE (12 g I2/100 g of oil) compared to IDTE (16 g I2/100 g of oil), which indicates the less risk of fuel polymerization (www.bdpedia.com). The density of the lipids extracted was found to be higher with DTE (0.8241 g/cm3) compared to IDTE (0.8012 g/cm3 with). With substrate variation, CWL evidenced its potential to accumulate lipids with good fuel properties and is in accordance to the prescribed standard requirements. CWL based lipid showed almost similar fuel characteristics (AN, 0.40 mg KOH/g of acid; IV, 11 g I2/100 g oil; density, 0.8342 g/cm3) as SDBM based fungal oil. 3.4. Degradation of substrate Substrate degradation (based on COD removal efficiency; initial, 40 g COD/l) improved with incubation time. With SDBM maximum degradation of 98.3% was observed at 96 h (Fig. 2). In the case of CWL, 60% of COD removal was observed at 48 h. Substrate degradation was observed along with lipid production. Fungi grow by utilizing the available nutrients present in CWL/SDBM through its metabolic activity and accumulate lipid granules. Variation in the lipid synthesis in the cytoplasm by fungi mainly dependent on the type of nutritional mode followed by the fungi and the availability of carbon and nutrients (Scandellari et al., 2009). Most of the fungi are chemo-heterotro/chemo-organotrophic which synthesizes the reserve food materials from pre-existing organic sources (simple sugars, amino acids, polysaccharides and proteins). Two-carbon acetyl-CoA molecules are the major components for lipid metabolism produced either by glycolysis process or by oxidative pentose phosphate pathway (OPPP) or by fatty acid catabolism. Similar to glycolysis, the OPPP is ubiquitous in fungi and play a major role in fatty acid synthesis (Fakas et al., 2009). Some of the evaluated previous studies the suitability of using wastewaters (olive oil mill) as a growth medium for oil-storing microorganisms such as Trichoderma viride (Domingues et al., 2000). Higher substrate degradation observed in the case of SDBM might be due to presence of relatively easy degradable substrate (glucose). The availability of substrate in CWL was either in aggregated [(waste solids) (hemicelluloses, lignin and lower amounts of cellulose residues)] or in dissolved state (nitrogen concentrations). For efficient growth and lipid accumulation, fungi depend on the
degradation of complex substrates. The extracellular enzymes of fungus degrade complex substrate and use them as metabolic source. Aspergillius sp. were used for the production of enzymes like glucoamylases, pectinases and galactosidases (Kang et al., 2004). The production of enzymes by fungi is specific towards the substrates and enhances the degradation of such complex compounds into simple compounds. This supports the mode of fungal nutrition (chemo-heterotrophic) thus leading to enhanced growth and lipid accumulation. 4. Conclusion Experimental data documented the feasibility of the isolated fungus strain Aspergillus sp. to produce biodiesel. Incubation time influenced both biomass growth and lipid productivity. Fungi grown in CWL showed a positive response with respect to biomass growth, lipid production and substrate degradation. Higher SFA fraction compared to USFA was found in both the substrates and in both extraction methods. The physical properties of fungal based biodiesel were in accordance with the biodiesel standards. Fuel obtained from fungal strain by employing cost effective experimental approach can serve as one of the alternative platform to compensate the raising future energy crisis. Acknowledgements The authors wish to thank Director, IICT for his support and encouragement in carrying out this work. GVS acknowledges Council for Scientific and Industrial Research (CSIR), New Delhi for providing research fellowship. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2011.06.084. References Aggelis, G., Komaitis, M., Papanikolaou, S., Papadopoulos, G., 1995. A mathematical model for the study of lipid accumulation in oleaginous microorganisms: lipid accumulation during growth of Mucor circinelloides CBS172-27 on a vegetable oil. Gracas y Aceites 46, 169–173. Alvarez, H.M., Steinbuchel, A., 2002. Triacylglycerols in prokaryotic microorganisms. Appl. Microbiol. Biotechnol. 60, 367–376. American Public Health Association(APHA), American water works Association (AWWA), 1998. Standard Methods for the Examination of Water and Wastewater. Water Environment Federation, Washington, DC. American Society for Testing and Materials. Standard specification for biodiesel fuel, 2002. (b100) blend stock for distillate fuels. Designation D6751-02. West Conshohocken (PA): ASTM International. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306.
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Dey, P., Banerjee, J., Maiti, M.K., 2011. Comparative lipid profiling of two endophytic fungal isolates – Colletotrichum sp. and Alternaria sp. having potential utilities as biodiesel feedstock. Bioresour. Technol. 102 (10), 5815–5823. Dionisi, F., Golay, P.A., Elli, M., Fay, L.B., 1999. Stability of cyclopropane and conjugated linoleic acids during fatty acid quantification in lactic acid bacteria. Lipids 34, 1107–1115. Domingues, F.C., Queiroz, J.A., Cabral, J.M.S., Fonseca, L.P., 2000. The influence of culture conditions on mycelial structure and cellulase production by Trichoderma reesei Rut C-30. Enzyme Microb. Technol. 26, 394–401. Fakas, S., Papanikolaou, S., Batsos, A., Galiotou-Panatoyou, M., Mallouchos, A., Aggelis, G., 2009. Evaluating renewable carbon sources as substrates for single cell oil production by Cunninghamella echinulata and Mortierella isabellina. Biomass Bioenergy 33, 573–580. Kang, S.W., Park, Y.S., Lee, J.S., Hong, S.I., Kim, S.W., 2004. Production of cellulases and hemicellulases by Aspergillus niger KK2 from lignocellulosic biomass. Bioresour. Technol. 91, 153–156. Kavadia, A., Komaitis, M., Chevalot, I., Blanchard, F., Marc, I., Aggelis, G., 2001. Lipid and gamma-linolenic acid accumulation in strains of Zygomycetes growing on glucose. J. Am. Oil Chem. Soc. 78, 341–346. Lewis, T., Nichols, P.D., McMeekin, T.A., 2000. Evaluation of extraction methods for recovery of fatty acids from lipid-producing microheterotrophs. J. Microbiol. Methods 43, 107–116. Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C., Yoken, R.H., 1995. Manual of Clinical Microbiology, sixth ed. American Society for Microbiology, Washington, DC.
Papanikolaou, S., Komaitis, M., Aggelis, G., 2004. Single cell oil (SCO) production by Mortierella isabellina grown on high-sugar content media. Bioresour. Technol. 95, 287–291. Scandellari, F., Hobbie, E.A., Ouimette, A.P., Stucker, V.K., 2009. Tracing metabolic pathways of lipid biosynthesis in ectomycorrhizal fungi from position-specific 13 C-labelling in glucose. Environ. Microbiol. 11, 3087–3095. Smedes, F., Askland, T.K., 1999. Revisiting the development of the Bligh and Dyer total lipid determination method. Mar. Pollut. Bull. 38, 193–201. Steinbu, C.A., Fu, C.B., 1998. Bacterial and other biological systems for polyester production. Trends Biotechnol. 16, 419–427. Venkata Mohan, S., Prathima Devi, M., Mohanakrishna, G., Amarnath, N., Lenin Babu, M., Sarma, P.N., 2011. Potential of mixed microalgae to harness biodiesel from ecological water-bodies with simultaneous treatment. Bioresour. Technol. 102, 1109–1117. Vicente, G., Bautista, L.F., Gutierrez, F.J., Rodrıguez, R., Martınez, V., RodrıguezFrometa, R.A., RuizVazquez, R.M., Martınez, S.T., Garre, V., 2010. Direct transformation of fungal biomass from submerged cultures into biodiesel. Energy Fuels 24, 3173–3178. Wahlen, B.D., Willis, R.M., Seefeldt, L.C., 2011. Biodiesel production by simultaneous extraction and conversion of total lipids from microalgae, cyanobacteria, and wild mixed-cultures. Bioresour. Technol. 102 (3), 2724–2730. (accessed 13.09.10). Zhao, X., Hu, C., Wu, S., Shen, H., Zhao, Z.K., 2011. Lipid production by Rhodosporidium toruloides Y4 using different substrate feeding strategies. J. Ind. Microbiol. Biotechnol. 38, 627–632.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Biodiesel production from isolated oleaginous fungi Aspergillus sp. using corncob waste liquor as a substrate G. Venkata Subhash, S. Venkata Mohan ⇑ Bioengineering and Environmental Centre (BEEC), Indian Institute of Chemical Technology (IICT), Hyderabad 500 607, India
a r t i c l e
i n f o
Article history: Received 27 April 2011 Received in revised form 22 June 2011 Accepted 23 June 2011 Available online 30 June 2011 Keywords: Transesterification Fatty acid methyl ester (FAME) Saturated fatty acid (SFA) Substrate degradation
a b s t r a c t The study documented the potential of isolated filamentous fungus Aspergillus sp. as whole cell biocatalyst for biodiesel production using Sabourauds dextrose broth medium (SDBM) and corncob waste liquor (CWL) as substrates. SDBM showed improvement in both biomass production (13.6 g dry weight/ 1000 ml) and lipid productivity (23.3%) with time. Lipid extraction was performed by direct (DTE) and indirect (IDTE) transesterification methods. DTE showed higher transesterification efficiency with broad spectrum of fatty acids profile over IDTE. CWL as substrate showed good lipid productivity (22.1%; 2 g dry biomass; 48 h) along with efficient substrate degradation. Lipids derived from both substrates depicted high fraction of saturated fatty acids than unsaturated ones. Physical characteristics of fungal based biodiesel correlated well with prescribed standards. CWL derived biodiesel showed relatively good fuel properties (acid number, 0.40 mg KOH/g of acid; iodine value, 11 g I2/100 g oil; density, 0.8342 g/cm3) than SDBM derived biodiesel. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Some of the microorganisms such as bacteria, microalgae, fungi, etc. are reported to be oleaginous which can accumulate significant amounts of lipids (characteristically similar to that of vegetable oils) with good fraction of saturated fatty acids (SFA) than unsaturated fatty acids (USFA) (Papanikolaou et al., 2004; Wahlen et al., 2011; Venkata Mohan et al., 2011). Microorganisms metabolically transform the external carbon into carbohydrate or hydrocarbon and then to lipids. Lipids are considered to be important storage compounds in the form of triacylglycerols (TAG) and esters. If the lipid content in the cell exceeds 20%, then microorganism can be called as oleaginous microorganism. Actinomycetes group under restricted growth conditions show higher lipid productivity (70%) and accumulate them intracellularly as TAGs (Chisti, 2007). Few bacteria are reported to accumulate complex lipid in the form of polyhydroxyalkanoates (Steinbu and Fu, 1998). Yeast strains such as Rhodosporidium sp., Rhodotorula sp., and Lipomyces sp. showed intracellular lipids accumulation as high as 70% of their biomass dry weight (Kavadia et al., 2001). Substrate feeding modes show marked influence on the lipid productivity of oleaginous yeast Rhodosporidium toruloides Y4 (Zhao et al., 2011). In fungi, the storage lipids usually contain TAG with good percentage of SFA during stationary phase within special organelles known as lipid granules (Aggelis et al., 1995). Lipid accumulation abilities ⇑ Corresponding author. Tel.: +91 40 27191664. E-mail address: [email protected] (S. Venkata Mohan). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.06.084
of two endophytic fungal isolates Colletotrichum sp. and Alternaria sp. grown under optimum and nutrient-stress conditions were studied by Dey et al. (2011). Lipid content varies according to the nature of microorganism (biocatalyst), culture conditions (such as temperature, pH, nutrients, culture time, etc.) and substrate (Alvarez and Steinbuchel, 2002; Papanikolaou et al., 2004). Lipid profiles could also be modified by genetic engineering of oleaginous microorganisms (Vicente et al., 2010). The major advantage of microbial derived lipids are their ability to use inexpensive organic materials as the substrate, no need for arable land for their growth, does not lead to the food vs fuel crisis and their inherent feasibility of upscaling with practical viability. In this communication we have evaluated the potential of isolated fungus Aspergillus sp. as whole cell biocatalyst/feedstock for the production of lipids using corncob waste liquor (CWL) as primary substrate. An attempt was also made to study the changes in composition of fatty acid methyl ester (FAME) after transesterification of fungal oil by indirect and direct methods. 2. Experimental methodology 2.1. Fungal biocatalyst Fungal strain was isolated from the marshy soil in our institute. One gram of soil was aseptically transferred to 10 ml of sterile distilled water in laminar air chamber. Dilutions of 10 4 and 10 5 were spread on potato dextrose agar medium (PDA; pH 5.5; Hi-media) and incubated (28 ± 2 °C) for 36 h. After incubation four
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fungal colonies were observed. Among them a single colony was isolated based on the morphological characteristics viz., color, texture and mycelium formation. The fungal isolate was stained (colony and spores) with Lactophenol Cotton Blue (Murray et al., 1995) and visualized as smears with epifluorescent microscope (Nikon Eclipse-80i) at 400 magnification (Supplementary Fig. 2). The images were captured on encompassed digital camera (YIM-smt, 5.5 mega pixels) using NIS-elements (D3.0) software. Lactophenol Cotton Blue and microscopic observations also confirmed the isolate belongs to Aspergillus sp. Further, this isolate was used as whole cell biocatalyst for lipid extraction. The pure fungal strain was sub-cultured onto PDA slants and incubated for growth (36 h) at 28 ± 2 °C prior to their usage in the experiments. 2.2. Corncob waste liquor (CWL) CWL that was obtained after acid (2% HCl, pH, 5) and thermal (121 °C for 45 min at 15 lbs pressure) treatment of corncob (COD, 5000 mg/l; VFA, 536 mg/l; TSS, 150 mg/l; reducing sugars, 882 mg/l; carbohydrates, 567.24 mg/l and color, 400 hazen units) was used as substrate for harnessing biodiesel. 2.3. Fungal Lipid production The detailed experimental methodology designed and executed as depicted in Supplementary Fig. 1. Aspergillus sp. was inoculated into 500 ml flasks containing 250 ml of sterile Sabourauds dextrose broth medium [SDBM; 40 g glucose, 10 g peptone in 1000 ml of water (pH 5.0)] and kept in a rotary shaker incubator (150 rpm) at 30 °C. Initially biomass and lipid productivity was optimized using SDBM culture. At regular 12 h time intervals (12, 24, 36, 48, 60, 72, 84 and 96 h) fungal biomass was collected by centrifuging the mycelium at 10000 rpm for 15 min and biomass growth (wet weight (g)) was estimated gravimetrically. Following biomass concentration, the cell precipitate was washed with distilled water (three times) and allowed to dry in oven at 60 °C for sufficient time. After drying, the dried biomass was crushed to powder using piston-motor and dry weight (g) was measured. The resulting dried fungal biomass acquired from different time intervals was used for lipid extraction. Two different types of transesterification procedures viz., single and two-step were employed and their efficiencies of extraction and conversion were evaluated. Based on the initial optimization experiments using SDBM, lipid production was further studied with the same isolate using CWL as substrate (5000 mg COD/l) and kept for growth under same optimum conditions obtained during preliminary/primary study (pH, 5; 30 °C, 150 rpm, 48 h incubation). The dried fungal biomass was subjected to lipid extraction. 2.3.1. Transesterification Two types of transesterification procedures were employed for the conversion of fungal lipids to fatty acid methyl esters (FAME) viz., direct transesterification of biomass (i.e. without the initial extraction step (single-step transesterification) and indirect transesterification by initial extraction of lipids from biomass followed by transesterification (two-step transesterification) (Vicente et al., 2010; Lewis et al., 2000). 2.3.1.1. Direct transesterification (DTE). Transesterification reaction mixture (methanol:hydrochloric acid:chloroform (10:1:1 (v/v/v); 12 ml) was added to dried fungal biomass (200 mg) in screw-cap test tube and subjected to sonication (5 min; 20 kHz) to facilitate cell membrane disruption (Lewis et al., 2000). Presonicated cells were suspended in the solution, vortexed and immediately placed at 90 °C under stirring (300 rpm) for 8 h in water bath. At the end of reaction, the mixture was diluted with distilled water (DW) and
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then extracted with ethyl acetate (EA; 2 100 ml). The EA layer was washed with DW to bring the solution pH to neutral. The two immiscible layers of DW and EA were separated using separating funnel and the solvent mixture was dried over anhydrous sodium sulfate and evaporated in a rotary evaporator. The residual FAME’s were weighed to calculate the yield and then analyzed to determine its composition. 2.3.1.2. Indirect (two-step) transesterification (IDTE). Dried fungal biomass (200 mg) in screw-cap tube was subjected to addition of each solvent in sequence (for 30 s) viz., chloroform, methanol, water ((1:2:0.8 (v/v/v)) and the resulting mixture was subjected to sonication (5 min; 20 kHz) for disruption of fungal cell wall. Biomass-solvent mixture after 18 h (with occasional shaking) was phase separated using a glass funnel. Total lipid extract (recovered from upper chloroform phase) was used for FAME preparation. Acid transesterification was done by adding the mixture of concentrated sulfuric acid–methanol (1 ml:100 ml/l of oil) to the preheated fungal oil and kept for stirring (200 rpm; 35 °C) for 1 h followed by 24 h incubation at room temperature (200 rpm). After incubation, the reaction mixture was allowed to stand for 1 h to separate the top layer with excess sulfuric acid (recover impurities) and the lower phase with oil. The lower oil layer is further subjected to alkali transesterification with sodium methoxide solution [sodium hydroxide (6.5 g/l of oil) in methanol (150 ml/l of oil)]. During reaction startup, half of the reaction mixture was added to the oil and kept for heating (55 °C; 200 rpm) for 1 h followed by cooling (room temperature; 24 h). After transesterification, the solvent was partially removed and extracted with EA (2 100 ml). After separating the top (mixture of FAME dissolved in EA) layer, the mixture was washed with DW for neutralization and the solvent was dried over anhydrous sodium sulfate followed by rotary evaporation. 2.4. Analysis The broth and CWL were evaluated through chemical oxygen demand (COD), pH and ORP analysis according to the procedures outlined in standard methods (APHA, 1998). The composition of concentrated FAME samples were detected by gas chromatography (GC; Shimadzu 17A) using flame ionization detector (FID), capillary column [SE 30 (30 0.32 mm)] and nitrogen as carrier gas (flow rate, 1 mL/min) [oven temperature; 140 °C (initially for 5 min), increased to 240 °C (ramp of 4 °C/10 min); injector temperature: 280 °C; detector temperatures 300 °C; split ratio: 1:10]. FAME composition was identified by comparing the retention time (RT) with authentic standards (FAME mix; SUPELCO; LB66766) and quality was compared with ASTM standards (ASTM, 2002). The quality of the fungal biodiesel was determined by analyzing iodine value (IV) (D 1959-97), acid number (D664) and density (ASTM; D6751-02). 3. Results and discussion 3.1. Biomass and lipid productivity with SDBM Fungal isolate used as whole cell biocatalyst showed good biomass growth with concomitant lipid accumulation by consuming available carbon and other nutritive compounds present in SDBM (Supplementary Fig. 3). Lipid accumulation showed a gradual increment up to 48 h followed with a drop which continued up to 96 h (Fig. 1). Fungal growth was relatively slow in between 0 and 12 h which also resulted in lower lipid accumulation. The wet/dry (W/D) biomass at this particular phase was 24/6 g with a lipid productivity of 10.2% (based on dry weight). A good
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Table 1). Both the methods showed higher fraction of SFA than USFA which is considered as a potential feature to indicate the fuel quality of fungal based diesel. DTE method evidenced good fatty acid profile comprising of 10 SFA and seven USFA in agreement to its high lipid productivity while IDTE owing to its low lipid productivity showed a moderate fatty acid profile depicting six SFA and three USFA. Presence of higher SFA indicates the conversion efficiency of the transesterification method to convert fatty acids into methyl esters. Though the amounts of fatty acids are less in IDTE method, the existence of less number of USFA indicates its negligible spin towards unsaturation. Marginal variation was observed between the SFA profile of both methods. DTE method additionally showed the presence of lauric acid methyl ester (C12:0), tridecanoic acid methyl ester (C13:0), palmitic acid methyl ester (C16:0) and behenic acid methyl ester (C22:0). Presence of additional SFA indicates the efficacy of DTE method than IDTE towards acquiring high fuel and lubricating properties. The higher yield of lipid profile with DTE method might be attributed to the improved amount of total lipids extracted from submerged culture (Lewis et al., 2000). This observation is supported by previous report depicting increased recovery of fatty acids from microorganisms by DTE method (Dionisi et al., 1999). Solvent addition strategy in sequence significantly influences the extraction efficiency of lipids. Adding solvents in the order chloroform–methanol–water showed better extraction (Smedes and Askland, 1999).
Fig. 1. Lipid profile in relation to dry biomass weight with the function of time.
increment in both biomass growth (W/D; 48/7.80 g, 60/8.8 g) and lipid productivity (12.42%, 18.34%) was obtained at 24 and 36 h, respectively. Maximum biomass growth (72/13.6 g (W/D)) and lipid productivity (23.33%) was registered at 48 h. Further increment in cycle period showed a gradual decrement in the lipid accumulation capability of fungal isolate [17.42% (60 h), 15.45% (72 h), 12.32% (96 h)] due to utilization of reserved food granules (including lipids) in absence of available carbon source. Growth of fungus attained stationary phase at 48 h. Under carbon deprived conditions over a prolonged period, the fungal hyphae undergoing a stationary growth phase which establishes balance between hyphal mass tending to either increment or decrement. Increment in lipid productivity was found to depend on combined influence of biomass growth and substrate availability.
3.2. Lipid productivity with CWL Alternatively, CWL was also evaluated as substrate (main carbon source) for fungal-lipid production. This approach facilitates utilization of waste for value-added product recovery with simultaneous treatment. In this case the optimum conditions obtained during primary study performed with SDBM were employed. Fungi showed effective biomass growth as well as lipid production with CWL. Lipid productivity of 22.1% (DTE method per dry biomass) was observed along with 15/2 g (W/D) of biomass growth at 48 h. Similar to SDBM, CWL based lipids also documented higher fraction of SFA compared to USFA (Table 1; Supplementary Table 1). On the contrary, CWL showed relatively good fatty acid profile compared to SDBM. SFA profile showed the presence of additional fatty acid (caprylic acid methyl ester (C8:0)) with CWL-lipid
3.1.1. Transesterification influence on FAME profile All the available microbial lipids are not suitable for production of biodiesel. Only lipids with fatty acid ester linkages (also referred to as saponifiable lipids) and free fatty acids can produce FAME, which can be used as biodiesel. During transesterification with methanol, both microbial lipids and free fatty acids can be converted to FAME in presence of a suitable acid catalyst. Indirect and direct transesterification methods were evaluated for the preparation of fungal based FAME. The proportions of SFA and USFA extracted from the fungal-lipid varied with the function of applied transesterification methods (Table 1; Supplementary
Table 1 Characterization of fungal lipid extracted from SDBM and CWL. Physical properties of biodiesel
Biodiesel standards*
<0.5 Acid number (mg KOH/g of acid) Iodine value <25 (g of I2/ 100 g of oil) Density (g/ 0.8800 cm3) Type of SFA (%, in relation to the total fatty acid) Type of USFA (%, in relation to the total fatty acid) *
ASTM (2002).
SDBM-lipid
CWL-lipid
Direct transesterification method (DTE)
Indirect transesterification method (IDTE)
Direct transesterification method (DTE)
0.42
0.47
0.40
16.0
12.0
11
0.8012
0.8241
0.8342
C10:0 (0.31), C12:0 (0.37), C13:0 (0.04), C14:0 (0.19), C15:0 (0.10), C16:0 (0.73), C17:0 (29.6), C18:0 (48.7), C20:0 (2.99), C22:0 (0.06)
C8:0 (2.96), C10:0 (2.4), C14:0 (0.70), C15:0 (0.28), C18:0 (56.6), C20:0 (1.12)
C8:0 (0.17), C10:0 (0.31), C12:0 (0.36), C13:0 (0.04), C14:0 (0.19), C15:0 (0.2), C16:0 (0.73), C17:0 (22.45), C18:0 (43.4), C20:0 (12.2), C22:0 (2.9)
C14:1n9c (0.1), C16:1n9c (14.4), C18:1n9t (1.60), C18:1n9c (0.11), C18:2n6c (0.42), C18:3n3 (0.09), C22:1n9 (0.089)
C16:1n9c (16.8), C18:2n6c (4.5), C22:1n9 (14.42)
C16:1n9c (14.8) C18:1n9t (0.42) C18:1n9c (0.6) C18:2n6c (0.42)
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compared to SDBM-lipid. While the USFA profile with CWL showed the presence of myristoleic acid methyl ester (C14:1n9c), linolenic acid methyl ester (C18:3n3) and erucic acid methyl ester (C22:1n9)). However, SDBM-lipid documented the presence of additional three USFA (myristoleic acid methyl ester (C14:1n9c), Cis-11-eicosenoic acid methyl ester (C20:1), erucic acid methyl ester (C22:1n9)). All the SFA and USFA detected with CWL-lipid were noticed to possess good lubrication and less unsaturation properties. Apart from the mentioned features the fatty acids derived also have potential and are used in making of pharmaceuticals and lubricating agents. Lipid productivity and fungal biomass growth obtained from this study documented the potential of Aspergillus sp. as whole cell biocatalyst to produce good quality biofuel utilizing CWL as substrate. Fig. 2. Substrate (COD) degradation profile during lipid production.
3.3. Fuel characterization of fungus lipids The lipids extracted from fungus by two transesterification methods evidenced good fuel characteristics but varied with the methods employed (Table 1). Acid number (AN), iodine value (IV) and density were in accordance with the biodiesel standards indicating the potential of fungal oil to use as biodiesel. DTE showed efficient fuel properties compared to IDTE. DTE method showed low AN (0.42 mg KOH/g of acid) compared to IDTE (0.47 mg KOH/g of acid). Low AN value indicates non-corrosive nature of the fuel. Similarly, IV of fungal oil was found to be low with DTE (12 g I2/100 g of oil) compared to IDTE (16 g I2/100 g of oil), which indicates the less risk of fuel polymerization (www.bdpedia.com). The density of the lipids extracted was found to be higher with DTE (0.8241 g/cm3) compared to IDTE (0.8012 g/cm3 with). With substrate variation, CWL evidenced its potential to accumulate lipids with good fuel properties and is in accordance to the prescribed standard requirements. CWL based lipid showed almost similar fuel characteristics (AN, 0.40 mg KOH/g of acid; IV, 11 g I2/100 g oil; density, 0.8342 g/cm3) as SDBM based fungal oil. 3.4. Degradation of substrate Substrate degradation (based on COD removal efficiency; initial, 40 g COD/l) improved with incubation time. With SDBM maximum degradation of 98.3% was observed at 96 h (Fig. 2). In the case of CWL, 60% of COD removal was observed at 48 h. Substrate degradation was observed along with lipid production. Fungi grow by utilizing the available nutrients present in CWL/SDBM through its metabolic activity and accumulate lipid granules. Variation in the lipid synthesis in the cytoplasm by fungi mainly dependent on the type of nutritional mode followed by the fungi and the availability of carbon and nutrients (Scandellari et al., 2009). Most of the fungi are chemo-heterotro/chemo-organotrophic which synthesizes the reserve food materials from pre-existing organic sources (simple sugars, amino acids, polysaccharides and proteins). Two-carbon acetyl-CoA molecules are the major components for lipid metabolism produced either by glycolysis process or by oxidative pentose phosphate pathway (OPPP) or by fatty acid catabolism. Similar to glycolysis, the OPPP is ubiquitous in fungi and play a major role in fatty acid synthesis (Fakas et al., 2009). Some of the evaluated previous studies the suitability of using wastewaters (olive oil mill) as a growth medium for oil-storing microorganisms such as Trichoderma viride (Domingues et al., 2000). Higher substrate degradation observed in the case of SDBM might be due to presence of relatively easy degradable substrate (glucose). The availability of substrate in CWL was either in aggregated [(waste solids) (hemicelluloses, lignin and lower amounts of cellulose residues)] or in dissolved state (nitrogen concentrations). For efficient growth and lipid accumulation, fungi depend on the
degradation of complex substrates. The extracellular enzymes of fungus degrade complex substrate and use them as metabolic source. Aspergillius sp. were used for the production of enzymes like glucoamylases, pectinases and galactosidases (Kang et al., 2004). The production of enzymes by fungi is specific towards the substrates and enhances the degradation of such complex compounds into simple compounds. This supports the mode of fungal nutrition (chemo-heterotrophic) thus leading to enhanced growth and lipid accumulation. 4. Conclusion Experimental data documented the feasibility of the isolated fungus strain Aspergillus sp. to produce biodiesel. Incubation time influenced both biomass growth and lipid productivity. Fungi grown in CWL showed a positive response with respect to biomass growth, lipid production and substrate degradation. Higher SFA fraction compared to USFA was found in both the substrates and in both extraction methods. The physical properties of fungal based biodiesel were in accordance with the biodiesel standards. Fuel obtained from fungal strain by employing cost effective experimental approach can serve as one of the alternative platform to compensate the raising future energy crisis. Acknowledgements The authors wish to thank Director, IICT for his support and encouragement in carrying out this work. GVS acknowledges Council for Scientific and Industrial Research (CSIR), New Delhi for providing research fellowship. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2011.06.084. References Aggelis, G., Komaitis, M., Papanikolaou, S., Papadopoulos, G., 1995. A mathematical model for the study of lipid accumulation in oleaginous microorganisms: lipid accumulation during growth of Mucor circinelloides CBS172-27 on a vegetable oil. Gracas y Aceites 46, 169–173. Alvarez, H.M., Steinbuchel, A., 2002. Triacylglycerols in prokaryotic microorganisms. Appl. Microbiol. Biotechnol. 60, 367–376. American Public Health Association(APHA), American water works Association (AWWA), 1998. Standard Methods for the Examination of Water and Wastewater. Water Environment Federation, Washington, DC. American Society for Testing and Materials. Standard specification for biodiesel fuel, 2002. (b100) blend stock for distillate fuels. Designation D6751-02. West Conshohocken (PA): ASTM International. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306.
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Dey, P., Banerjee, J., Maiti, M.K., 2011. Comparative lipid profiling of two endophytic fungal isolates – Colletotrichum sp. and Alternaria sp. having potential utilities as biodiesel feedstock. Bioresour. Technol. 102 (10), 5815–5823. Dionisi, F., Golay, P.A., Elli, M., Fay, L.B., 1999. Stability of cyclopropane and conjugated linoleic acids during fatty acid quantification in lactic acid bacteria. Lipids 34, 1107–1115. Domingues, F.C., Queiroz, J.A., Cabral, J.M.S., Fonseca, L.P., 2000. The influence of culture conditions on mycelial structure and cellulase production by Trichoderma reesei Rut C-30. Enzyme Microb. Technol. 26, 394–401. Fakas, S., Papanikolaou, S., Batsos, A., Galiotou-Panatoyou, M., Mallouchos, A., Aggelis, G., 2009. Evaluating renewable carbon sources as substrates for single cell oil production by Cunninghamella echinulata and Mortierella isabellina. Biomass Bioenergy 33, 573–580. Kang, S.W., Park, Y.S., Lee, J.S., Hong, S.I., Kim, S.W., 2004. Production of cellulases and hemicellulases by Aspergillus niger KK2 from lignocellulosic biomass. Bioresour. Technol. 91, 153–156. Kavadia, A., Komaitis, M., Chevalot, I., Blanchard, F., Marc, I., Aggelis, G., 2001. Lipid and gamma-linolenic acid accumulation in strains of Zygomycetes growing on glucose. J. Am. Oil Chem. Soc. 78, 341–346. Lewis, T., Nichols, P.D., McMeekin, T.A., 2000. Evaluation of extraction methods for recovery of fatty acids from lipid-producing microheterotrophs. J. Microbiol. Methods 43, 107–116. Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C., Yoken, R.H., 1995. Manual of Clinical Microbiology, sixth ed. American Society for Microbiology, Washington, DC.
Papanikolaou, S., Komaitis, M., Aggelis, G., 2004. Single cell oil (SCO) production by Mortierella isabellina grown on high-sugar content media. Bioresour. Technol. 95, 287–291. Scandellari, F., Hobbie, E.A., Ouimette, A.P., Stucker, V.K., 2009. Tracing metabolic pathways of lipid biosynthesis in ectomycorrhizal fungi from position-specific 13 C-labelling in glucose. Environ. Microbiol. 11, 3087–3095. Smedes, F., Askland, T.K., 1999. Revisiting the development of the Bligh and Dyer total lipid determination method. Mar. Pollut. Bull. 38, 193–201. Steinbu, C.A., Fu, C.B., 1998. Bacterial and other biological systems for polyester production. Trends Biotechnol. 16, 419–427. Venkata Mohan, S., Prathima Devi, M., Mohanakrishna, G., Amarnath, N., Lenin Babu, M., Sarma, P.N., 2011. Potential of mixed microalgae to harness biodiesel from ecological water-bodies with simultaneous treatment. Bioresour. Technol. 102, 1109–1117. Vicente, G., Bautista, L.F., Gutierrez, F.J., Rodrıguez, R., Martınez, V., RodrıguezFrometa, R.A., RuizVazquez, R.M., Martınez, S.T., Garre, V., 2010. Direct transformation of fungal biomass from submerged cultures into biodiesel. Energy Fuels 24, 3173–3178. Wahlen, B.D., Willis, R.M., Seefeldt, L.C., 2011. Biodiesel production by simultaneous extraction and conversion of total lipids from microalgae, cyanobacteria, and wild mixed-cultures. Bioresour. Technol. 102 (3), 2724–2730.