Accepted Manuscript Biosynthesis, characterization and enzymatic transesterification of single cell oil of Mucor circinelloides – A sustainable pathway for biofuel production Ana K.F. Carvalho, Juan D. Rivaldi, Jayne C. Barbosa, Heizir F. de Castro PII: DOI: Reference:

S0960-8524(15)00019-X http://dx.doi.org/10.1016/j.biortech.2014.12.110 BITE 14440

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

10 November 2014 18 December 2014 19 December 2014

Please cite this article as: Carvalho, A.K.F., Rivaldi, J.D., Barbosa, J.C., de Castro, H.F., Biosynthesis, characterization and enzymatic transesterification of single cell oil of Mucor circinelloides – A sustainable pathway for biofuel production, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2014.12.110

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Biosynthesis, characterization and enzymatic transesterification of single cell oil of Mucor circinelloides – A sustainable pathway for biofuel production

Ana K. F. Carvalho, Juan D. Rivaldi, Jayne C. Barbosa, Heizir F. de Castro* Engineering School of Lorena, University of São Paulo 12602-810, Lorena, São Paulo–Brazil

*Corresponding author Engineering School of Lorena, University of São Paulo, Laboratory of Biocatalysis, Estrada Municipal do Campinho, 12602-810, Lorena-SP, Brazil. Tel.: +55 12 31595149 E-mail address: [email protected] (H.F. de Castro).

Abstract The filamentous fungus Mucor circinelloides URM 4182 was tested to determine its ability to produce single-cell oil suitable for obtaining biodiesel. Cell growth and lipid accumulation were investigated in a medium containing glucose as the main carbon source. A microwaveassisted ethanol extraction technique (microwave power ≤ 200 W, 50-60°C) was established and applied to lipid extraction from the fungal hyphae to obtain high lipid concentration (44% wt) of the dry biomass, which was considerably higher than the quantity obtained by classical solvent methods. The lipid profile showed a considerable amount of oleic acid (39.3% wt), palmitic acid (22.2 %wt) and γ-linoleic acid (10.8 % wt). Biodiesel was produced by transesterification of the single-cell oil with ethanol using a immobilized lipase from Candida antarctica (Novozym® 435) as the catalyst. 1H-NMR and HPLC analyses confirmed conversion of 93% of the single-cell oil from M. circinelloides into ethyl esters (FAEE).

Key-words: single-cell oil, Mucor circinelloides, microwave-assisted extraction, lipid, biodiesel

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1. Introduction The first generation of biodiesel is currently produced by the transesterification of triglycerides derived from vegetable oils and alcohols (methanol or ethanol) using alkaline catalysts. The limited availability and high cost of the traditional sources of lipid have motivated searches for new natural sources for the transesterification process. Single-cell oil has received attention as an alternative to traditional feedstocks to produce a third-generation biodiesel (Ho et al., 2014; Leiva-Candia et al., 2014). Lipids can be accumulated by microorganisms including filamentous fungi, yeast, bacteria and algae (Khot et al., 2014; Santamauro et al., 2014; Zhang et al., 2014; Da Rós et al., 2013). The production of biodiesel from algae and cyanobacteria is an important research topic, and the development of largescale production processes has already started in many countries. The production of lipids from filamentous fungi and yeast as feedstock for biofuels is not well explored. Single-cell oil is a natural lipid source, and its production is not affected by climate changes, location, harvest time and transport. These factors have an effect on the global cost of biodiesel produced from vegetable oil (Ratledge, 2004). The composition of microbial lipids is similar to vegetable oil and animal fats, and the profile of fatty acids (FAs) includes lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0), estearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3) and docohexeanoic acid (C22:6) (Khot et al., 2014; Vicente et al.; 2009). One of the most promising organisms for lipid production is the zygomycete fungus Mucor circinelloides. The fungus is rich in polyunsaturated fatty acids and γ-linolenic acid (GLA, 18:3, n-6), a valuable omega-6 fatty acid that is found mostly in plant-based oils (Vicente et al., 2010; Xia et al., 2011). Efficient extraction techniques are required to improve the competitiveness of microbial lipid production processes. Various extraction techniques including solvent extraction are used to isolate biocompounds from natural sources. However, these solvent3

based extraction methods are environmentally unsafe, time-consuming, and difficult to control and can cause undesired changes in the extractable compounds (Cravotto et al., 2008). Microwave-assisted ethanol extraction has been used to obtain essential oil from plants (Cravotto et al., 2008) and represents a potential method for extraction of lipids from microorganisms (Loong and Idris, 2014). This method is based on the absorption of electromagnetic energy by the solvents and cells and its transformation to heat energy. The irradiation provides selective heating of the sample-solvent mixture with low or imperceptible changes in lipid composition. In many cases, the microwave heat may be less damaging than any other extraction methods that include heating (Carrapiso and García, 2000). The advantages of this method include fast and high quantity lipid recovery and low solvent volume (Mercer and Armenta, 2011). The transesterification process for biodiesel production requires homogeneous and strong base catalysts such as sodium or potassium hydroxide. Heterogeneous catalysts such as enzymes immobilized in inert supports have received attention recently because of their high protein stability, easy recovery, and ability to be used in several reaction cycles (Christopher et al., 2014; Zhang et al., 2012). In this study, the synthesis of single-cell oil from Mucor circinelloides and its extraction using ethanol and microwave radiation were investigated. A comparison of the extraction yields with other extraction methods using solvent was also conducted. The lipid profile and oil properties were analyzed to determine its applicability as feedstock for biodiesel. The single-cell oil was converted into biodiesel with an enzyme-based heterogeneous catalyst, and its properties were analyzed according to the standard specifications.

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2. Material and Methods 2.1 Chemicals and reagents Palm (Elaeais guineensis) oil was acquired from AGROPALMA (Belém, PA, Brazil), and jatropha (Jatropha curcas) oil was obtained from Instituto Agronômico do Paraná– IAPAR (Londrina, PR, Brazil). All the chemicals and reagents were purchased from SigmaAldrich and used without any further purification. 2.2. Microorganism and culture conditions The fungus Mucor circillenoides f. griseo-cyanus URM-4182 was tested for its ability to produce lipids suitable as a material for biodiesel production. This strain has a documented high lipase activity (Andrade et al., 2014) and was purchased from the URM culture collection of Federal University of Pernambuco (Recife, PE, Brazil). The strain was maintained in potato-dextrose agar at 4°C and transferred each month. The cultivation was performed in 250-mL Erlenmeyer flasks containing 100 mL of medium with the following composition: glucose, 20 g/L; thiamine, 0.001 g/L; glutamic acid, 1.5 g/L; nicotinic acid, 0.001 g/L; ammonium sulfate, 1.5 g/L; and yeast extract, 0.5 g/L. The initial pH of the medium was adjusted to 4.5 by the addition of 1.0 mol/L NaOH. The culture was inoculated by aseptically transferring an aliquot of spore suspension to achieve a final concentration of 105 spores/mL. The medium was incubated at 26°C with agitation at 250 rpm for 120 h. The culture pH was measured every 24 h and manually adjusted to pH 4.5 by the addition of 1 M NaOH (Vicente et al, 2010). All of the experiments were performed in triplicate. 2.3 Lipid extraction The total lipids were extracted using a microwave-assisted ethanol extraction and the solvent extraction method as described by Folch et al. (1957) and Bligh and Dyer (1959). The 5

microwave-assisted extraction was conducted with 2.0 g of fungal biomass (94%, water) in a spherical glass vessel containing 25 mL of ethanol (96%) heated in a microwave reactor. The microwave reactor (Model Discover/University-Wave, Cem Corporation) consisted of a cylindrical internal chamber of 750 mm3 powered with compressed air and equipped with a magnetic stirrer and infrared sensors for temperature control. The operating temperature (50 or 60°C) was controlled automatically by variation in the microwave power with a maximum of 200 W. The extraction was performed in three steps of 10, 30 or 60 minutes each. After each extraction step, the suspension was filtered and the residual pellet was resuspended in the same volume of ethanol for the next step. The extract containing lipids was recovered and evaporated in a vacuum rotary evaporator, and the microbial oil was subsequently dried at 60°C until it reached a constant weight. The extraction with the Folch’s method was performed with 1.0 g of dried biomass in a glass vessel containing methanol and chloroform at a 2:1 (v/v) ratio. The extraction was assisted by sonication using a UP200S Ultrasonic Processor (Dr. Hielscher GmbH, Germany) in three steps of 10 min each (pulse mode 0.5 and 100% amplitude). The method of Bligh and Dyer was performed in a 100-mL Erlenmeyer flask containing 1.0 g of the dried biomass and 20 mL of a mixture containing chloroform, methanol and water at a 1:2:0.8 (v/v) ratio and glass beads (1:1, beads to biomass). The mixture was stirred (100 rpm) at room temperature for 18 h. The total lipids extracted by each technique were measured gravimetrically after the complete removal of the organic solvent by evaporation. 2.4 Enzymatic transesterification Biodiesel synthesis from the single-cell oil of M. circinelloides was performed in a 50mL cylindrical glass vessel containing the microbial oil and anhydrous ethanol at a molar ratio of 1:12. The reaction used immobilized lipase from Candida antarctica Novozym® 435 as a 6

biocatalyst at concentration of 10% of the total reaction mixture weight and iso-octane as the reaction solvent. The reaction was performed at 60°C with constant stirring (500 rpm) for a maximum period of 72 h (Da Rós et al, 2012). The biodiesel prepared with palm and jatropha oils under the same conditions was used as the control. 2.6 Biodiesel purification After the transesterification reaction, the immobilized lipase was separated by centrifugation (1520 x g) and the organic phase was washed twice with one volume of distilled water to remove any remaining ethanol and glycerol by-product. The residual ethanol and water were removed in a vacuum rotary evaporator, and the fatty acid ethyl esters were analyzed with the international standard specifications for biodiesel. 2.7 Analysis 2.7.1 Scanning electron microscopy (SEM) High-resolution scanning electron microscopy (LEO Model 1450 VP, ZEISS) was used to analyze the morphology of M. circinelloides mycelium after the microwave extraction. 2.7.2 Fatty acid composition An analysis of the fatty acid composition was performed with capillary gas chromatography (Agilent 6850 Series GC System) according to AOCS procedure 2-66 (AOCS, 2004). The chromatography equipment was equipped with a DB-23 Agilent capillary column (50% cyanopropyl-methylpolysiloxane) using helium as the carrier gas at a rate of 1.0 mL/min. The temperatures of the column, detector and injector were set at 215, 280 and

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250°C, respectively. The volume of the sample injection was 1.0 µL. All of the analyses were performed in triplicate. 2.7.3 Proton nuclear magnetic resonance spectrometry (1H NMR) The conversion of microbial oil into FAEEs was analyzed by NMR in a Mercury 300 MHz Varian spectrometer. FAEEs were dissolved in deuterated chloroform (CDCl3) using 0.3% tetramethylsilane (TMS) as the internal standard. The yield of microbial oil conversion into esters was determined according to the method proposed by Paiva et al. (2013). This method allows the identification of ester molecules produced during transesterification, which causes peaks in the region of 4.05 to 4.35 ppm. According to Equation 1, the signal of the ethoxyl hydrogen atoms of the ethyl esters split a quartet.

% EE = [(AC4 x 8)/(Add+ee)] x 100

(1)

where AC4 = area of the component fourth peak (quart) Add+ee = area of all signals between 4.35 and 4.05 % EE = yield of fatty ethyl esters (FAEEs)

In this equation, the Ac4 was obtained from the integration of the peak at 4.08 ppm. The area obtained corresponds to 1/8 of the whole ethoxi-carbon hydrogen area (-OCH2), whose signal appears in the region ranging from 4.05 to 4.20 ppm. The region near 4.08 ppm is the only region where crossover does not occur, and this integrated signal can be assigned to ethyl esters. 2.7.4. Mono-, Di- and Triglyceride analysis The glyceride analysis was performed in an Agilent 1200 Series liquid chromatograph (Agilent Technologies, USA) equipped with an Evaporative Light Scattering Detector and 8

Gemini C-18 (5 µm, 150 x 4.6 mm, 110 Å) column at 40°C. The mobile phase contained a mixture of acetonitrile (80%) and methanol (20%) at a flow rate of 1 mL/min for 6 min, 1.5 mL/min until 30 min and 3.0 mL/min until 35 min. All of the samples were dissolved in ethyl acetate-hexane (1:1, v/v), filtered through 0.22-µm membrane filters (Millipore) and injected in a volume of 10 µL. All the solvents were of HPLC grade, and the assays were performed at least in duplicate (Andrade et al, 2014). 2.7.5. Physicochemical properties The absolute viscosity of all the oil and biodiesel was determined with an LVDV-II cone and plate spindle Brookfield viscosimeter (Brookfield Viscometers Ltd., England) using a CP 42 cone. A circulating water bath was used to maintain the temperature of the samples at 40ºC. The shear stress measurements were taken as a function of shear rate, and the dynamic viscosity was determined as a slope constant. We used 0.5-mL samples, and the measurements were repeated three times. The density of biodiesel was determined with a DMA 35N EX digital densimeter (Anton Paar, USA) at 20ºC. We also used 2.0-mL biodiesel samples, and the measurements were replicated three times. The cetane number (CN), degree of unsaturation (DU), long-chain saturated factor (LCSF), saponification value (SV) and iodine value (IV) were empirically determined using BiodieselAnalyzer® software (version 1.1) as described by Talebi et al. (2014). 3. Results and Discussion 3.1 Production of single-cell oil The production of single-cell oil by M. circinelloides URM4182 was observed during the cultivation in the medium formulated with glucose as the main carbon source. The profiles of growth, glucose consumption and lipid production by M. circillenoides are shown in Fig.1. 9

The biomass concentration increased rapidly in the first 24 h of cultivation and achieved the stationary phase at 72 h. The highest biomass concentration (4.23 g/L), YX/S (0.44 g/g glucose) and QX (1.06g/L/d) were obtained at 96 h of cultivation, and the maximum lipid concentration was 44.0% with a volumetric lipid production rate (QP) of 0. 47 g/L/d (Table 1). The glucose was slowly consumed and it was not totally consumed (about 30% of the initial concentration remained in the medium) at the end of cultivation (120 h of growth). This behavior could be attributed to the insufficient pH control which makes use of intermittent addition of NaOH 1 mol/L at 24 h intervals. Additionally, low oxygen availability in the flask could affect substrate consumption. However, there are several factors affecting lipid synthesis in microorganisms including strain, medium and environmental factors (i.e., pH, temperature, stirring and dissolved oxygen) (El-Fadaly et al., 2009; Xia et al., 2011). The results achieved in this study were similar to findings observed with the cultivation of M. circillenoides ATCC1216B in a medium formulated with glucose as the main carbon source (Xia et al., 2011). In another study, biomass and single-cell oil were produced by the growth of a genetically modified M. circinelloides MU241, which produced 4.2 g/L biomass concentration and total oil of 22.9% dry mass (Vicente et al., 2010). The authors reported that the lipid concentration varies considerably during the first 48 h of growth (exponential growth phase) and becomes stabilized when cells reach the stationary phase. The lipid-accumulating fungi Mortierella isabellina achieved a lipid content of 67% growing in a medium based on glucose and 50.9% for medium containing xylose as the main carbon source (Zheng et al., 2012). The growth of this strain in non-detoxified hydrolyzate of wheat straw yielded lipid concentrations higher than 39.4%. Most reports in the literature regarding microbial oil production show lipid concentration values in the range of 20% to 50%.

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3.2 Microbial oil extraction The effect of different extraction techniques for recovering fatty acids from M. circinelloides was examined. The content of microbial oil was assayed by gravimetric estimation. Because the contents of proteins, pigments and other than non-lipid compounds in the extract can be considered low, all of the liquid obtained after ethanol evaporation can be counted as total microbial oil. The amount of M. circinelloides oil obtained using different extraction methods is shown in Table 2. The total lipid concentration extracted using the ethanol and microwave method is shown in Fig.2. The maximum lipid concentration was 41.8% (dry-biomass), which was achieved after three consecutive extraction steps at 60°C for 30 min each. However, the microbial oil obtained under these conditions was only 4% higher than the lipid attained with extraction steps of 10 min at the same temperature. Additionally, similar results were verified for extraction at 50°C. SEM microphotography of hyphae of M. circinelloides obtained before and after microwave-assisted lipid extraction with ethanol is shown in the Supplementary Material (Fig S1). Most fungi including M. circinelloides grow as hyphae, which have a cylindrical form and contain lipids particles and other cytoplasmic organelles. In this study, the electromagnetic radiation promoted the crushing of hyphae and we observed small and welldispersed hyphal fragments (Fig.S1 a - b). It is known that absorption of microwave energy in biological material is dependent on dipolar rotation and ionic conduction (Datta et al., 2005; Orsat et al., 2005). Molecules such as water and ethanol exhibit strong dipole moments that can be manipulated in alternating electromagnetic fields, which results in random collisions of these molecules and leads to thermal agitation and heating (Datta et al., 2005; Kaufmann and Christen, 2002). Furthermore, the electric field created in the microwave reactor accelerates 11

the oppositely charged ions in different directions and promotes collisions between the particles to increase the temperature of the material (Kaufmann and Christen, 2002; Orsat et al., 2005). SEM microphotography also revealed that some fungal hyphae remained unchanged after microwave treatment. However, the overall morphology of the pellets (S1-b) suggests that the microwave effectively disrupts cells for lipid extraction by ethanol. The comparison of total microbial oil attained by three methods showed that the microwave led to the best extraction of microbial oil (Table 2). The cavitation phenomenon produced by ultrasonic waves disrupts the cells in Folch’s method and allows for the extraction of microbial oil (21.7%,w/w). However, the concentration was 50% lower than we obtained with the microwave-assisted method. The extraction with the Bligh and Dyer method was less efficient compared with the other methods tested (7.0%,w/w). The glass beads used in this method were ineffective for cell disruption, which was attributed to the low numbers of bead impacts against mycelium (high solvent volume to biomass weight ratio). Iverson et al. (2001) indicated that natural samples containing lipid concentrations higher than 2% (w/w) could be underestimated by the Bligh and Dyer method due to the low solubility of non-polar lipids (triacylglycerols) in solvents that are relatively polar (chloroform, methanol and water). Vicente et al. (2009) reported a maximal oil concentration of 19.9% (w/w) when dried biomass of M. circinelloides was submitted to three different methods for microbial oil extraction. The methods included mixtures of chloroform/methanol, chloroform/methanol/ water, and n-hexane. Other studies described the utilization of solvent for lipid extraction. However, the modifications applied to the different methods (Bligh and Dyer, and Folch) are unspecified, which makes the evaluation and comparison of the results difficult. Therefore, ethanol extraction associated with magnetic energy could provide a clean and sustainable method for microbial oil extraction.

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3.3. Single-cell oil composition Biodiesel quality is associated with the raw material composition. Properties including fatty acid composition, chain length, degree of unsaturation, acidity index and iodine number are important parameters determining the potential of the microbial oil for biodiesel production. The characterization of the single-cell oil from M. circinelloides is shown in Table 3. Single-cell oil from M. circinelloides revealed fatty acids of carbon chain lengths ranging from 6 to 24. The most abundant fatty acids were oleic acid – C18:1; palmitic acid – C16:0; and linoleic acid – C18:2 . Other fatty acids (C8:0, C10:0, C12:0, C14:0, C16:0, C17:0, C18:0, C20:0, C22:1 and C24) represented less than 8% of the total fatty acid content. The high amount of saturated (35.8 wt.%) and monounsaturated fatty acids (41.3 wt.%), such as C16:0 and C18:1, shows the potential use of this oil for biodiesel production and other industrial applications. This profile has similar characteristics to vegetable oils conventionally employed for biodiesel production (Carvalho et al., 2013). Linoleic acid – C18:2 and γlinolenic acid (GLA) – C18:3 (10.7wt.%) are the polyunsaturated fatty acids (PUFAs) with the highest concentrations (Table 3). These compounds are highly desirable because they have applications for food, pharmaceutical and cosmetic formulation. Vicente et al. (2009) reported oil production from glucose by M. circinelloides with high concentrations of palmitic acid (20.0 wt.%), oleic acid C18:1 (37.0 wt.% ) and γ-linolenic acid C18:3 (18.5 wt.%). In another study of the growth of Aspergillus terreus on hydrocarbon substrate, fatty acids with chain lengths of C14 to C18 were predominant in the lipid profile and the PUFAs (C15:4, C17:4, C19:4, C32:3, and C33:4) were approximately 9% (wt.%) (Kumar et al., 2010). Some other properties of the oil used for biodiesel production have also significant influence on the biofuel characteristics. The iodine value (85 g I2/100 g oil) of the microbial oil produced by M. circinelloides indicated a high degree of saponifiable lipids, including 13

triglycerides, diglycerides, monoglycerides and free fatty acids. This value is lower than the normal established value. Furthermore, we observed a high degree of unsaturation (82.46 %) that could generate methyl and ethyl esters with low thermal-oxidative stability. This characteristic is desirable because there is an inverse relationship between the oxidative stability of the lipids and the hydrodynamic property (fluidity) of the biofuels produced from certain oil. Thus, the high degree of unsaturation of the biosynthesized oil indicates that it is suitable for the production of biodiesel for combustion at low temperature because it has a low risk of solidification. In addition to the melting point, the number of unsaturated carbonic chains of the lipids used for biofuel production affects the cetane number, combustion heat (measurement of energetic power) and boiling point of biodiesel (Canakci et al., 1999; Vicente et al., 2009). Polyunsaturated fatty acids (PUFAs) with four or more double bonds are usually undesirable for biodiesel production and were not identified in the single-cell oil obtained from M. circinelloides. A comparison of the microbial oil from M. circinelloides and palm oil shows that both oils have similar oleic acid and monounsaturated fatty acid compositions (Table 3). In general, oil from filamentous fungi differs from vegetable oil in the high concentration of PUFAs, which are compounds that are especially important for obtaining intermediate molecules for chemical, pharmaceutical and food industries (Khot et al., 2012; Vicente et al., 2009). We also observed that there was approximately 50% less polyunsaturated lipids in single-cell oil from M. circinelloides than in the non-edible jatropha oil, which is another important source for biodiesel production (Table 3). 3.4 Biodiesel production The single-cell oil obtained from M. circinelloides was subjected to transesterification using ethanol as an acyl acceptor and Candida antarctica lipase B immobilized on 14

macroporous acrylic resin as the catalyst following methodology previously established for microbial oil (Da Rós et al, 2012). The conversion of the microbial oil and purity of the biodiesel was assessed by nuclear magnetic resonance spectroscopy (1H NMR) and liquid chromatography (HPLC) analysis. NMR is extensively used to monitor the transesterification process and quality of biodiesel. This analysis is based on the variation of glycerol methylene and ester ethoxy hydrogens in the region of 4.05 - 4.35 ppm during the transesterification reaction (Paiva et al., 2013). The 1H NMR spectrum of single-cell oil and fatty acid ethyl esters (FAEE) of M. circinelloides are shown in Supplementary Material (Fig S2). The spectrum shows a peak at 4.08 ppm, corresponding to methylene protons of the ethanolic fraction of ethyl esters. Table 4 shows the areas of the peak corresponding to ethyl esters and the percentage conversion of the single-cell oil. Botn experiments reached yields of around 93% after 72 h reaction. The absence of peaks (multiples) in the 4.20 ppm region of the biodiesel spectrum that correspond to the protons of the 1 and 3 carbon of glycerol in the triglyceride molecules confirms the high conversion of the microbial oil. The chromatography analysis confirmed this high rate of conversion ̴ 93% ± 0.4 of the single-cell oil of M. circinelloides into biodiesel (Table 5). The amount of monoglycerides and diglycerides found were 4.2% and 2.8% , respectively. The ASTM standard for biodiesel (ASTM D6751) establishes values up to 0.5% for mono and diglycerides. Generally, the presence of unreacted glycerides reduces the quality of the final product because these compounds increase the turbidity and viscosity of the product. The presence of the glycerides and lower conversion of the oil could be associated with the high concentration of C18:3, which is a polyunsaturated fatty acid with a long carbon chain. The catalyst (immobilized lipase) is more efficient for the conversion of saturated fatty acids with short carbon chains (Christopher et al., 2014). 15

Further removal of these impurities can assure the standard quality of the biodiesel from M. circinelloides. Furthermore, the low concentration of these unreacted glycerides and the absence of triglycerides in biodiesel from M. circinelloides show the efficiency of conversion of triglycerides into ethyl esters promoted by the immobilized lipase. The characteristics of the FAEEs obtained from M. circinelloides were compared to FAEEs produced from palm and jatropha oil (Table 5), which are raw materials used extensively by the biodiesel industry in Brazil. A comparison of the biodiesel obtained under the same conditions from jatropha and palm oil shows yields slightly higher than the yields obtained using the single-cell oil from M. circinelloides. These results are comparable to data reported in the literature for biodiesel produced from vegetable and microbial oils. Vicente et al. (2009) reported the extraction and conversion of oil from M. circinelloides into fatty acid methyl esters (FAMEs) by a homogeneous acid catalyst (BF3; HCl and H2SO4). The authors achieved yields ranging from 91.5 to 98.0%. The authors also described a one-step (direct) production of biodiesel from biomass of M. circinelloides using the same conditions for producing biodiesel with yields higher than 99.0% (Vicente et al., 2009). In a previous work, B. cepacia lipase immobilized on SiO2-PVA was used at a fixed proportion of 500 units per gram of palm and jatropha oil to catalyze biodiesel synthesis. This approach resulted in FAEEs yields of 99.6 and 98.5%, respectively (Carvalho et al., 2013). The biodiesel quality is dependent on the fatty acid profile of the oil used as feedstock for its production. In addition to the molecular characteristics of the oils, properties such as cetane number (CN), kinematic viscosity, density and cold filter plugging point (CFPP) are parameters widely used to characterize biodiesel. The CN is a measurement of the combustion quality during ignition of biodiesel and depends on the distribution of saturated and unsaturated fatty acids or esters. The ignition quality is associated with the interval of ignition. A fuel with high CN number has a short 16

ignition interval and starts to burn shortly after it is injected into an engine. In this study, CN was estimated at 49.2, 59.3 and 54.2 for blends of ethyl esters from jatropha, palm and M. circinelloides oil, respectively (Table 5). These values are in compliance with the standards for biodiesel (ASTM D6751), which recommend a minimum CN of 47. The European minimum CN number is 51, and all of the biodiesel complies with the specifications except for biodiesel from palm oil. Generally, high CN values are observed for saturated fatty esters and increase with chain length (Varatharajan and Cheralathan, 2012). CN values greater than the minimum specified by the engine manufacturer do not necessarily increase the engine performance. This value is associated not only with the molecular composition of lipid used for biofuel production but also with the engine design and characteristics (Knothe et al., 2005). In addition to cetane number, viscosity is another parameter that affects biodiesel quality. The kinematic viscosity is dependent on the degree of unsaturation and chain length of the fatty acid blends or alcohol moiety that compounds the biodiesel. Viscosity increases with molecular weight but decreases with increasing level of unsaturation and temperature (Knothe and Steidley, 2005). A higher viscosity results in low volatility and poor atomization of the fuel during the injection, which promotes an incomplete burning and carbon deposits in the injector and combustion chamber (Knothe et al., 2005; Ramos et al., 2009). In the present study, the kinematic viscosity obtained for biodiesel from M. circinelloides was slightly higher than the limit established by the standard (1.9 - 6.0 mm2/s). The biodiesel samples from jatropha and palm are within the standard range (Table 4). The cold filter plugging point (CFPP) is the lowest temperature at which a given volume of fuel passes through a standard filter in a specified time with specific cooling conditions. Similar to the CN, the CFPP of biodiesel mainly depends on the saturated fatty acid composition (Knothe and Steidley, 2005; Ramos et al., 2009). This value is especially 17

important in cold temperature countries where a high CFPP could clog engine filters. The CFPP for biodiesel from M. circinelloides was estimated at 3.9°C and is similar to values observed for biodiesel from jatropha and palm oil (Table 4). These values are in compliance with the Brazilian biodiesel standard that establishes a maximum CFPP of 5.0°C for colder regions of the country (ANP). The standard specification of CFPP is dependent on the season and location. In other countries using UNE-EN 14214 or ASTM D6751, various temperature limits are specified for different times of the year depending on climate conditions (Knothe, 2006). 4. Conclusions Single-cell oil from M. circinelloides was analyzed as a potential feedstock for biodiesel production. The total oil obtained was 44% of dried biomass after 96 h of cultivation. Microwave-assisted ethanol extraction is a fast and sustainable method for recovering high lipid concentrations from fungi. The lipid profile of M. circinelloides showed high concentrations of saturated and monounsaturated fatty acids, predominantly C16:0 and C18:1. The enzymatic transesterification of the single-cell oil with ethanol achieved yields of ethyl esters of 93%. The transesterification of the single-cell oil from M. circinelloides is a suitable approach to completely fulfill the standard specifications for renewable diesel. Acknowledgements The authors gratefully acknowledge the financial support of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Juan D. Rivaldi would especially like to thank the support of CAPES (Process PNPD 02565/09-9). References 18

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Figure captions

Figure 1. Profile of biomass production (○), glucose concentration (■) and lipids (▲) by M. circinelloides URM 4182 at 26 °C and 250 rpm. Figure 2. Single-cell oil obtained by microwave-assisted ethanol extraction from M. circinelloides URM 4182.

22

20 40 15 30 10 20 5

Lipids (% w/w)

Glucose - Biomass (g/L)

50

10

0

0 0

24

48

72

96

120

Time (h)

Figure 1

23

Total lipids ( %, w/w)

50 45

50°C 60°C

40 35 30 25 20 15 10 5 0 10

30

60

Time (min)

Figure 2

24

Table 1 – Biochemical and kinetic parameters of batch cultivation of M. circinelloides URM 4182 using glucose as a carbon source in 96 h.

Parameter

Value

Lipid concentration, P (g/L)

1.86±0.01

Biomass, X (g/L)

4.23±0.09

Substrate consumption, S (g/L)

9.82±0.10

Specific yield of lipid, YP/X (g lipid/g biomass)

0.44±0.15

Process product yield, YP/S (g lipid/g substrate)

0.19±0.24

Microbial biomass yield, YX/S (g biomass/g substrate)

0.43±0.05

Volumetric lipid production rate, QP (g/L/day)

0.47±0.06

Volumetric substrate consumption rate, QS (g substrate/L/day)

2.46±0.21

Volumetric biomass production rate, QX (g biomass/L/day)

1.06±0.02

Specific rate of lipid production, qP (g lipid /g biomass/day)

0.11±0.03

Specific rate of substrate consumption, qS (g substrate/g biomass/ day)

0.58±0.05

values represent the mean of tripilicate experiments.

25

Table 2 – Total lipid concentrations from M.circillenoides URM 4182 as a function of different extraction methods.

Extraction Method

Lipids (g)

Dried biomass (g)

Total lipids (%,w/w)

Microwave-assisted ethanol extraction

0.84±0.02

2.02±0.01

41.80±0.04

Folch

0.22±0.01

1.05±0.03

21.70±0.06

Bligh and Dyer

0.07±0.05

1.03±0.06

7.00±0.05

26

Table 3 – Properties of single-cell oil from Mucor circinelloides URM 4182 after 96 h of cultivation in medium containing glucose. Oil Property 2

Kinematic viscosity at 40°C (mm /s) Acidic value (mg KOH/g oil) Saponification value (mg KOH/g oil) Iodine value (g I2/100 g oil) Fatty acids composition (wt %) Caproic C6:0 Caprilic C8:0 Capric C10:0 Lauric C12:0 Miristic C14:0 Pentadecanoic acid C15:0 Palmitic C16:0 Palmitoleic C16:1 Margaric C17:0 Cis-10-heptadecenoic C17:1 Estearic C18:0 Trans elaidic C18:1 Oleic C18:1 Trans t-linoleic C18:2 Linoleic C18:2 Gamma linolenic C18:3 Linolenic C18:3 Arachidonic C20:0 Eicosenoic C20:1 Behenic C22:0 Lignoceric C24:0 Saturated (wt.%) Monounsaturated (wt.%) Polyunsaturated (2,3) (wt.%) Degree of unsaturation (%) a

Jatrophaa

Palma

34.5 0.3 141 101

36.8 0.3 198 98

M.circinelloides (this work) 36.2 38.8 203 85

0.1 12.9 5.6 -

1.2 46.8 3.8 -

0.3 0.1 0.3 2.1 2.1 0.2 22.2 1.0 0.4 0.2 7.7 0.3

39.8 40.0 -

37.6 10.5 -

39.3 0.2 9.5 10.7 0.1 0.4 0.7 0.5 1.7

18.6 39.8 40.2

51.9 37.6 10.5

35.8 41.3 20.5

120.2

58.6

82.5

Carvalho et al. 2013

27

Table 4 - Area of the integration peaks and the conversion percentage of single-cell oil of M. circinelloides to ethyl esters determined by 1H NMR

Transesterification reaction

Area AC4

Area Add+ee

Yield (%)

T1

0.11

0.94

93.5

T2

0.10

0.86

92.9

28

Table 5 – Biodiesel characteristics obtained from transesterification of vegetable (jatropha and palm) and single-cell (M. circinelloides ) oils using ethanol as acyl acceptor.

Jatropha curcas

Palm

SCO

Ester content (%)a

96.1±0.4

98.0±0.6

93.0±0.4

Monoglycerides (%)

3.2±0.2

2.0±0.1

4.2±0.2

Diglycerides (%)

0.8±0.1

0

2.8±0.1

0

0

0

Density at 20°C (kg/m )

883±0.5

874±1.6

878±1.9

Cetane Number Kinematic viscosity at 40°C (mm2/s) Cold filter plugging point (CFPP, °C) Degree of unsaturation (%)

49.2

59.3

54.2

4.8±0.5

5.5±0.3

6.2±0.2

2.4

4.2

3.9

120.2

58.6

82.5

Triglycerides (%) 3

a

Total ethyl ester formed with respect to the initial oil mass in the reaction medium, i.e., YGlobal =[(biodiesel mass)/(oil mass)] × 100.

29

Graphical abstract

30

Highlights

1. A Brazilian Mucor circinelloides strain was found to be a good SCO producer. 2. The microwave-assisted EtOH extraction led to the best extraction of SCO. 3. The SCO showed considerable amount of monounsaturated fatty acids. 4. The enzymatic ethanolysis of the SCO achieved high yields of esters. 5. Biodiesel properties from SCO are comparable with those from vegetable oils.

31