Biochemical Engineering Journal 48 (2009) 2227

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Biochemical Engineering Journal

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Biodiesel production from biomass of an oleaginous fungus

Gemma Vicente a, , L. Fernando Bautista a , Rosala Rodrguez a , F. Javier Gutirrez a , Irantzu Sdaba a , Rosa M. Ruiz-Vzquez b , Santiago Torres-Martnez b , Victoriano Garre b
a b

Department of Chemical and Environmental Technology, ESCET, Universidad Rey Juan Carlos, C/ Tulipn s/n, 28933 Mstoles, Madrid, Spain Departamento de Gentica y Microbiologa (Unidad asociada al IQFR-CSIC), Facultad de Biologa, Universidad de Murcia, 30071 Murcia, Spain

a r t i c l e

i n f o

a b s t r a c t
The present paper introduces the lamentous fungus Mucor circinelloides as a potential feedstock for biodiesel production. These microbial lipids showed a high content (>85%) of saponiable matter and a suitable fatty acid prole for biodiesel production. The effectiveness of the lipid extraction process was studied for three different solvent systems: chloroform:methanol, chloroform:methanol:water and n-hexane. Biodiesel was produced by acid-catalysed transesterication/esterication following two different approaches: transformation of extracted microbial lipids and direct transformation of dry microbial biomass. After 8 h of reaction at 65 C in the presence of BF3 , H2 SO4 or HCl as acid catalysts, the direct process produced fatty acid methyl esters (FAMEs) with higher purities (>99% for all catalysts) than those from the two-step process (91.498.0%). In addition, the yield was also signicantly higher in the direct transformation due to a more efcient lipid extraction when the acid catalyst was present. 2009 Elsevier B.V. All rights reserved.

Article history: Received 19 January 2009 Received in revised form 9 July 2009 Accepted 22 July 2009

Keywords: Biodiesel Fatty acid methyl esters Microorganism Fungi Mucor circinelloides Microbial oil

1. Introduction Demand for fatty acid methyl esters (FAMEs) as diesel fuel (biodiesel) has increased signicantly due to the instability of petroleum prices and the development of government measures in many countries around the world that establish a minimum proportion of biofuel for all petrol and diesel used in transport. For instance, the European Union establishes a minimum content of 5.75% of biofuel by 2010 (European Union Directive 2003/30/EC) and the United States plans to increase the amount of bioethanol and biodiesel to 12.95 and 36 billion gallons by 2010 and 2022, respectively (Energy Independence and Security Act of 2007). Biodiesel constitutes a renewable fuel that is compatible with current commercial diesel engines and has clear benets relative to diesel fuel including enhanced biodegradation, reduced toxicity and a lower emission prole [1]. Nonetheless, biodiesel presents some disadvantages. One of its drawbacks is the high manufacturing cost, which is mainly due to the high cost of the vegetable oil. Actually, 7090% of the biodiesel production cost corresponds to raw vegetable oil. In addition, the biodiesel industry competes with the food industry for oil crops. In fact, it has been calculated that a very large percentage of the current available arable land is required to achieve the current biofuel objectives using crops such

Corresponding author. Tel.: +34 91 4888531; fax: +34 91 4887068. E-mail address: gemma.vicente@urjc.es (G. Vicente). 1369-703X/$ see front matter 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2009.07.014

as rapeseed or sunower. Therefore, it is necessary to explore new raw materials that reduce the biodiesel price without competing with food production. In this context, oils from microorganisms (also called single-cell oils) constitute a promising alternative for producing biodiesel since they present many advantages over vegetable oils from oleaginous plants. Microorganisms can accumulate high level of lipids and do not require arable land. In addition, the production of these microorganisms does not compete with food production since biomass residuals can be used as carbon source. Microorganisms which accumulated more than 2025% lipids are usually referred to as oleaginous species [2]. In most cases, the oil from these microorganisms is in the form of triglycerides, which are also the main component in vegetable oils and animal fats. Therefore, the microbial lipids can potentially be used as raw material for biodiesel production using the common way to produce FAMEs in the biodiesel industry, i.e. the transesterication reaction with methanol in the presence of a basic catalyst (e.g. sodium and potassium hydroxide or sodium methoxide). However, the utilisation of these catalysts in the transesterication of vegetable oils or animal fats with a high concentration of free fatty acids produces soaps by neutralisation, which in turn partially consumes the catalyst, decreases the biodiesel yield and complicates the separation and purication steps [1]. Free fatty acids are usually present in the lipid composition of microbial cells [3]. However, soap formation from free fatty acid neutralisation can be avoided by using an acid catalyst such as sulphuric or hydrochloric acids. The acids catalyse the free fatty acid esterication with methanol to also produce FAMEs, increasing the biodiesel yield [4].

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The principal oleaginous microbial species are microalgae, bacteria, fungi and yeasts. The use of microorganisms as a source of lipids has been extensively investigated for their application as food additives, pharmaceuticals and feed ingredients for aquaculture [2,3,510]. Microorganisms are sources of edible oils because they have the ability to produce oils rich in polyunsaturated fatty acids, which are in demand as dietary supplements and for infant nutrition [7]. More recently, some works have dealt with the use of oleaginous microorganisms for biodiesel production. Particularly, microalgae, which capture carbon dioxide by transformation into lipids using sunlight, have attracted recent attention and investment for biofuel production because of their higher oil productivity and faster growth compared to conventional energy crops [11,12]. However, these photosynthetic microorganisms have problems associated with their growth in bioreactor systems due to the necessity of light supply and large acreages. In addition, the economics of producing biodiesel from microalgae need to improve to make it competitive with diesel [11]. Through changing culture conditions or using genetic engineering modications, some autotrophic microalgae can be converted to heterotrophic microalgae and such heterotrophic microalgae can also accumulate oils using organic carbon as the carbon source instead of CO2 [13]. According to Miao and Wu [14], heterotrophic growth of a microalga (Chlorella protothecoides) results in higher biomass production and higher lipid accumulation in cells in comparison to the autotrophic growth of this microalga. These authors reported an integrated method for biodiesel production from heterotrophic C. protothecoides oil by acidic transesterication. Conversely, little information has been reported so far on the use of lipids from yeast, fungi and bacteria for biodiesel production. Nonetheless, two recent reviews dealt with the related research about these oleaginous microorganisms, and the prospects of such microbial oils for biodiesel production [13,15]. In comparison to the microalgae, the growth of these microorganisms can be carried out in conventional microbial bioreactors, which will improve the biomass yield and will reduce the cost of biomass and oil productions. In this context, one work described the acid methanolysis of biomass from two yeasts (Lipomyces starkeyi and Rhodosporidium toruloides) and one lamentous fungus (Mortirella isabellina) for biodiesel production [16]. In the present work, we have investigated the production of biodiesel from the fungal Mucor circinelloides. This fungus shows many relevant features favouring its use for biodiesel production, including a high level of lipids in the mycelium (around 25% dry mass in wild-type strains) [7], good biomass production during submerged batch cultivation in bioreactors using a wide range of carbon sources [17], and a proven capacity to grow in large industrial stirred-tank fermenters (220 m3 ) to produce oil rich in -linolenic acid [10]. More signicantly, the regulation of lipid accumulation in this fungus has been extensively studied [18], and key genes have been identied that could be manipulated using a large number of already available molecular tools, including gene silencing (RNAi) [19]. Based on its potential use in biofuel production, the Department of Energy of the United States (DOE) has selected this fungus to sequence its genome through the bioenergy program at the Joint Genome Institute, a project that is nearly complete. The present study included the lipid extraction and characterisation, in addition to the biodiesel production from M. circinelloides biomass. Two procedures for biodiesel production were compared: the extraction of lipids from M. circinelloides biomass followed by the transformation of the extracted lipids into FAMEs and the direct conversion of the M. circinelloides biomass, without previous extraction, to produce FAMEs. The high quality of the biodiesel produced using the direct method, which complies with the European and American standards, suggests that M.

circinelloides biomass could be used as a feedstock to produce biodiesel. 2. Materials and methods 2.1. Strain and growth conditions The strain MU241, derived from R7B [20] after replacement of its leuA mutant allele by a wild-type allele, was used as a wildtype strain to produce fungal biomass. For biomass production, 2.5 105 spores per plate (9.5 cm diameter) were inoculated on solid minimal medium pH 4.5 (YNB; [21]) with a cellophane sheet and incubated for three days at 26 C in the presence of white light (4.8 W m2 ). Mycelia grown on the cellophane sheet were harvested and dried between paper towels, frozen in liquid nitrogen, lyophilised, weighed to estimate dry mass and grounded using mortar and pestle. 2.2. Chemicals Extraction solvents, chloroform (>99%), methanol (>99%) and n-hexane (>96%), were purchased from Scharlab (Madrid, Spain). Reagent grade 35% hydrochloric acid, 14% boron triuoride solution in methanol (both purchased from SigmaAldrich, Madrid, Spain) and analytical grade 9597% sulphuric acid (provided by Scharlab) were used as catalysts. Commercial rened sunower oil was used for the control reactions. All other chemicals were reagent grade or higher. Type I deionised water for standards was produced by a MilliQ-gradient system (Millipore, Billerica, MA, USA). 2.3. Analytical methods Characterisation of microbial lipids was performed following standard methods when possible. However, due to the limited amount of microbial biomass, some analytical procedures were adapted from existing standard methods but in other cases, alternative analytical methods were selected. Iodine number was calculated as described in EN 14214:2003 standard. Acid value of sunower oil was measured according to EN 14104:2003 standard. Free fatty acid content in the lipid fraction extracted from the microorganisms was measured following a colorimetric procedure [22] based on the formation of cupric soaps and further quantication of the chromophore complex by absorbance at 715 nm in a Cary 500 spectrophotometer (Varian Inc., Palo Alto, CA, USA). Fatty acid proles of both vegetable and microbial oils were performed by gas chromatography in a CP-3800 gas chromatograph (Varian Inc.) tted with FID detector and TRB-FFAP capillary column (60 m length, 0.32 mm internal diameter; 0.25 m lm thickness. Teknokroma, Barcelona, Spain). Prior to GC analysis, the oil samples were transformed into their corresponding methyl esters by saponication in 0.5 M KOH in methanol solution (30 min at 90 C) followed by treatment with 14% boron triuoride in methanol (10 min at 90 C) and extraction with n-hexane/water. Finally, 3 l of the organic phase containing FAMEs was injected into the capillary column where the separation was achieved using a temperature ramp (1 C min1 ) from 150 C to 240 C at a ow rate of 1 ml min1 (injector temperature: 180 C, detector temperature: 280 C, injection mode: splitless). Identication of chromatographic peaks was performed by comparison with a fatty acid methyl ester standard mixture (Reference 07131-1AM, Supelco, Bellefonte, PA, USA) and quantication by means of external standards and their corresponding calibration curve. Phosphorous content in microbial oil was determined by inductively coupled plasma-optical emission spectrometry (ICP-OES)

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using a Vista AX model (Varian Inc.). The analysis was performed according to ASTM D-5185-05 standard. Free fatty acids, triglycerides, diglycerides, monoglycerides, fatty acid methyl esters (FAMEs), carotenoids, sterol esters, sterols and tocoferols, retinoids and polar lipids were identied and quantied by TLC analysis in both vegetable and microbial oil and biodiesel. Chromatographic separation was developed in 20 cm 20 cm silica-coated aluminium plates (Alugram Sil G/UV. Macherey-Nagel GmbH, Dren, Germany) using a solvent mixture of 88 vol% n-hexane, 11 vol% diethyl ether and 1 vol% glacial acetic acid. Visualisation was carried out by staining with iodine. Digital image analyses of staining plates were performed with Un-ScanIt Gel 6.1 software (Silk Scientic Inc., Orem, UT, USA) and the lipid compositions were quantied by the corresponding calibration curves. 2.4. Extraction of lipids Lipids from lyophilised and grinded microbial biomass were extracted using three different solvent systems: chloroform:methanol (C:M), chloroform:methanol:water (C:M:W) and n-hexane. Lipid extraction with C:M system (2:1, volume ratio) [23] was performed using, approximately, 200 mg of dried biomass. Three washes with 10 ml, 5 ml and 5 ml of solvent mixture, respectively, were carried out for 10 min each together with ultrasonication to favour cell membrane disruption. The solvent mixture containing extracted lipids was separated from residual biomass by centrifugation and all the fractions from each stage were pooled and the solvent removed in a rotary evaporator. Washed residual biomass was dried and weighted to calculate the biomass free of lipids and while extraction losses were estimated as mass difference between lipophilised raw microorganism and the sum of extracted lipids plus biomass free of lipids. The next extraction method [24] consisted on a monophasic extraction using C:M:W (1:4:0.8, volume ratio) following partition of organic and aqueous phases by adding chloroform and water to reach a solvent ratio of 1:1:0.9. After separation of the organic phase containing the microbial lipids, the aqueous phase was washed three times with chloroform in a separatory funnel. All the organic phases were mixed and the solvent removed in a rotary evaporator as previously described. Extraction using n-hexane was carried out following a similar method than that used with the C:M system above. For all methods above, the extraction yield (wt%) relative to dry biomass weight was measured and the reported values correspond to the average of three replicates. 2.5. Biodiesel production Experiments were planned to determine the effect of three different acid catalysts (namely BF3 , H2 SO4 and HCl) and the reaction temperature (25 C and 65 C). Reactions of extracted microbial lipids were performed in 15 ml glass closed vessels with magnetical stirring (900 rpm) using a methanol to oil molar ratio of 60:1 and a catalyst concentration of 8 wt% relative to microbial oil. The reactor was then immersed in a thermostatic bath at the reaction temperature for 8 h. The
Table 1 Lipid extracted from M. circinelloides using three different solvent mixtures. Extraction solvents Chloroform:methanol Chloroform:methanol:water n-Hexane Lipid content (wt%) 19.9 1.3 19.0 1.1 15.3 1.0

FAME layer was collected and the crude glycerol was washed ve times with n-hexane:diethyl ether (80:20) and the same volume of water. The upper organic layers were put together with the rst FAME layer and the solvent was removed in a rotary evaporator leaving the residue containing the FAMEs, which was used in measuring the reaction yield relative to the dry microbial biomass and in quality characterisation by TLC. For comparison purposes, reaction control experiments of a mixture of 72% rened sunower oil and 28% free fatty acids were carried out following the same method than described for reaction of microbial oil extracted from M. circinelloides biomass. The free fatty acids added were prepared by complete saponication of rened sunower oil with 4% KOH in ethanol solution and further acid treatment with 6 M H2 SO4 to yield the free fatty acid after the corresponding purication by aqueous washing. Direct transformation of dry microbial biomass was carried out according to the method of Lewis et al. [25] in a single step in the same reactor than that used in the reaction of extracted oil. In this direct process, a methanol:chloroform 10:1 (v/v) mixture was used as a reagent-solvent system where the appropriate amount of the corresponding acid catalyst was dissolved. After 8 h in the reaction vessel immersed in a thermostatic bath, the FAMEs were obtained and characterised as described for the two-step process above. Three replicates of each reaction experiment were performed and their average results were given. 3. Results and discussion 3.1. Microbial biomass production and lipid extraction To produce biodiesel, M. circinelloides biomass was obtained from the prototrophic strain MU241 grown on a solid minimal medium containing glucose as a carbon source (10 g l1 ). After three days of growth, a 3.73 0.27 g l1 of fungal dry mass was obtained. Lipids were extracted from this fungal biomass using three mixtures of solvents: chloroform:methanol (C:M), chloroform:methanol:water (C:M:W) and n-hexane. Table 1 shows the lipid content, the biomass free of lipids and, therefore, the extraction losses for all the extraction procedures. Both mixtures with chloroform and methanol (C:M and C:M:W) lead to the highest quantity of extracted lipids (19.9 wt% and 19 wt%, respectively). Traditionally the binary mixture of chloroform and methanol (2:1 v/v) [23] and the use of ternary solvent systems C:M:W [24] have been considered simple and rapid methods for the extraction and purication of lipids from biological materials [26]. The second method (C:M:W) involves a substantial decrease in the volume of solvent used in the extraction procedure in comparison with the C:M system [25]. In this work, however, the binary mixture C:M leads to the highest values of extracted lipids (19.9 wt%) and the lowest valued of extraction losses (1.7 wt%). Therefore, it was the chosen extraction procedure. The third extraction method with n-hexane [14,27] was carried out to avoid the use of chlorinated solvents, because of the adverse effect of these solvents on the environment. Many solvents have been studied to replace the chloroform in the lipid extraction [28], n-hexane being an interesting alternative which provides efcient lipid extraction [14]. However, its use as an extraction solvent led to the lowest quantity of extracted lipids (15.3 wt%) in this

Biomass free of lipids (wt%) 78.4 1.0 76.5 0.6 80.9 2.0

Extraction losses (wt%) 1.7 0.3 4.5 1.6 3.8 3.0

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work, because extracted M. circinelloides lipids also contained polar lipids. The amounts of extracted lipids from M. circinelloides were compared with those reported for oleaginous seeds (sunower, rapeseed and soybean crops) [29]. The lipid amounts in the fungal biomass seemed to be lower (1519 wt%) than the oil extracted from rapeseed and sunower seeds (35 wt% and 40 wt%) and similar to the oil extracted from soybean seeds (18 wt%). However, the values for the vegetable crops refer only to the oil extracted from the seeds, and therefore, those values decrease drastically when considering the entire plant weight. In fact, this weight should always be taken into account in order to make a valid comparison. 3.2. Lipid characterisation Not all lipids obtained from microbial biomass are suitable for making biodiesel. Only lipids with fatty acid ester linkages (also referred to as saponiable lipids) and free fatty acids can produce FAMEs, which can be used as biodiesel if they comply with the current existing standards (ASTM D 6751 in the United States or EN 14213 and 14214 in the European Union). As conventional vegetable oils, microbial saponiable lipids and free fatty acids can be converted into FAMEs through a transesterication and esterication reaction with methanol, respectively, in the presence of a suitable catalyst. The saponiable lipids and free fatty acids were 86.15% of the total lipids extracted from the M. circinelloides biomass, with free fatty acids, polar lipids (phospholipids, sphingolipids and saccharolipids) and triglycerides as the main components (Table 2). The non-saponiable lipid fraction consisted of small amounts of carotenoids, sterols, tocopherols and retinoids (Table 2). The high concentration of free fatty acids (31.6 1.3%) in M. circinelloides determines that an acid-catalysed process is more suitable for producing biodiesel than an alkali one in order to avoid yield losses from free fatty acid neutralisation [30]. Table 3 shows the free fatty acid prole for the saponiable lipids and free fatty acids extracted from M. circinelloides fungus. Microbial oils usually differ from most vegetable oils in being quite rich in polyunsaturated fatty acids [11]. The high degree of unsaturation inherent to the FAMEs derived from these fatty acids would evidence lower oxidative stability, but excellent fuel properties at low temperatures, which is an advantage in winter operation [1]. The content of polyunsaturated fatty acids with four or more double bonds was absent in the M. circinelloides oil, but it contained 14.3 0.6 and 18.5 0.7% of linoleic (two double bonds) and linolenic (three double bonds) acids, respectively. Nevertheless, M. circinelloides oil also contained monounsaturated fatty acids and saturated fatty acids in relatively high concentrations. In fact, this fungus contained large amounts of oleic acid (37 1%) and
Table 2 Composition of the lipids extracted from M. circinelloides. Lipid classication Type of lipid Free fatty acids Triglycerides Diglycerides Monoglycerides Phospholipids Sphingolipids and saccharolipids Sterol esters Total saponiable lipids Carotenoids Sterols and tocopherols Retinoids Total non-saponiable lipids Concentration (wt%) 31.60 1.30 13.70 1.80 3.20 0.50 1.60 0.20 20.80 1.90 15.50 1.90 0.09 0.02 86.50 6.40 1.30 0.09 0.04 7.00 0.30 13.50

Table 3 Fatty acid composition and iodine value in the saponiable lipids and fatty acids extracted from M. circinelloides. Fatty acid Lauric acid Myristic acid Myristoleic acid Pentadecanoic acid Palmitic acid Palmitoleic acid Stearic acid Oleic acid Linoleic acid Linolenic acid Arachidic acid Gadoleic acid Behenic acid Erucic acid Lignoceric acid Nervonic acid Other Iodine value (g I2 /100 g) 12:0 14:0 14:1 15:0 16:0 16:1 18:0 18:1 18:2 18:3 20:0 20:1 22:0 22:1 24:0 24:1 Concentration (wt%) n.d 0.8 0.1 n.d n.d 20 1 2.3 0.4 21 37 1 14.3 0.6 18.5 0.7 0.20 0.02 0.59 0.03 0.5 0.1 n.d 1.2 0.3 n.d 2.0 0.9 107.6

palmitic acid (20 1%). The iodine value is a measure of the unsaturation level and, therefore, only depends on the oil used as the raw material. Thus, the calculated iodine value for the saponiable lipids and the free fatty acids extracted from M. circinelloides was 107.6 mg I2 g1 , being far below the specied limit of 120 mg I2 g1 in the European Union Standards. 3.3. Biodiesel production from microbial oil Transesterication/esterication reactions using extracted microbial oil were conducted at two different temperatures with each acid catalyst. Fig. 1 shows the yield of FAMEs on the basis of the dry weight biomass. At 25 C, regardless of the catalyst, the yield of FAMEs was approximately constant, achieving values between 14.1% and 14.6%. Although the catalytic reactions conducted at 65 C with BF3 and H2 SO4 showed similar yields (13% and 14%, respectively), the results obtained with HCl were lower (10.4%), likely due to the gaseous nature of this acid. The composition analyses of the biodiesel produced at 25 C after 8 h of reaction showed that, in all cases, the ester content was lower than that included in the EN 14214 standard (96.5%) (Table 4). In addition, triglyceride content for all catalysts and free fatty acid content for H2 SO4 slightly surpassed their corresponding specications, indicating that these reaction conditions are not suitable for

Free fatty acids and saponiable lipids

Non-saponiable lipids

Fig. 1. Effect of temperature and type of catalyst on FAMEs yield.

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Table 4 Quality control of the biodiesel obtained in the two-step process of transformation of M. circinelloides. Property 25 C BF3 Ester content (wt%) Monoglyceride content (wt%) Diglyceride content (wt%) Triglyceride content (wt%) Free glycerol (wt%) Total glycerol (wt%) Acid value (mg KOH g1 ) Non-saponiable lipids (wt%) Polar lipids (wt%)
a

65 C H2 SO4 88.8 n.da n.da 0.8 0.0035 0.8035 0.8 9.9 0.1 HCl 91.9 n.da n.da 1.1 0.0030 1.1030 n.da 6.8 0.2 BF3 98.0 n.da n.da n.da 0.0020 0.0020 n.da 2.0 n.da H2 SO4 91.5 n.da 0.1 0.1 0.0030 0.2030 0.6 8.0 n.da HCl 95.8 n.da n.da 0.1 0.0030 0.1030 0.2 4.0 n.da

EU Standard EN 14214

US Standard ASTM D6751

90.7 n.da n.da 1.2 0.0022 1.2022 0.2 7.9 0.1

96.5 min. 0.8 max. 0.2 max. 0.2 max. 0.02 max. 0.25 max. 0.5 max. n.sa n.sa

n.sa n.sa n.sa n.sa 0.02 max. 0.24 max. 0.5 max. n.sa n.sa

n.d = not detected; n.s = not specied limit.

M. circinelloides lipid transformation. However, the above results from reactions of microbial oil are in contrast to the control reactions carried out with the high free fatty acid sunower oil, where the FAME purities achieved were about 80% in reactions performed in the same conditions and with the same catalysts, since a signicant fraction of triglycerides (>50%) were not transformed. The monoglyceride, diglyceride, non-saponiable lipid and polar lipid contents were negligible in all cases. In the case of microbial oil, triglyceride conversion was >90% in all systems. This higher conversion of triglycerides observed in microbial oil could be due to its higher content in short chain length fatty acids (Table 3), which are more reactive through acid transesterication reactions than long ones. The free glycerol content was lower than the European and American standard limits, indicating that the glycerol residuals were eliminated during the purication treatment. Besides, nonsaponiable lipids were signicant in the M. circinelloides-derived biodiesel obtained with the three catalysts, which means that these types of lipids were not eliminated during the purication stage. However, they are not considered in the biodiesel specications established so far. The biodiesel obtained also had small quantities of polar lipids, which were lower than 0.2% in all cases (Table 4) since the conversions of these types of lipid were >99%. These compounds are residuals of non-converted polar lipids and they are not considered in the current biodiesel specications. The reactions carried out at 65 C showed a signicant increase in FAME purity for all catalytic processes (Table 4). However, the content of FAMEs fullled the European standard only when BF3 was used as the catalyst. The content of free fatty acids, glycerides, free glycerol and total glycerol reached values corresponding to both standards, except for the free fatty acid content using H2 SO4 , which yielded a value slightly higher than these specications. This implies that the transesterication and esterication reactions were completed and that the glycerol was eliminated during the purication treatment. At 65 C, triglyceride conversions with all catalysts in both microbial oil and sunower oil experiments were

>99%. Non-saponiable lipids were between 2.0% and 8.0% and, therefore, they were lower than the corresponding ones obtained at 25 C. In this sense, the degradation of these types of lipids is favoured at high temperatures, facilitating their elimination during the purication stage. By contrast, the conversions of polar lipids were nearly 100% since they were not detected in the biodiesel obtained with the three catalysts. Although reaction at 65 C in the presence of BF3 produced FAMEs that complied with biodiesel standards, a method that combines the lipid extraction and the acid-catalysed transesterication/esterication of the extracted lipids in one step was carried out [25]. In this process, methanol and chloroform were also used as solvents with a methanol/chloroform ratio of 10:1 (v/v) and H2 SO4 , HCl or BF3 as the acid catalyst. In reactions taking place at 65 C during 8 h, biodiesel yields were 18.5%, 18.0% and 17.5% relative to the dry mass of M. circinelloides, using H2 SO4 , HCl and BF3 , respectively. In comparison to the process with a previous extraction stage (Fig. 1), the yields obtained were signicantly higher. These yields were even higher than the corresponding theoretical yield calculated for this microorganism (15.3%), indicating that this direct transformation strategy improved the amount of total lipids extracted compared with the conventional methods for lipid extraction from microorganisms [23,24]. These high yields could be a consequence of the extraction-reaction medium being highly acidic, which causes signicant cell disruption [25]. The quality of the biodiesel produced in the one-step procedure was also compared with the corresponding specied biodiesel limits in standards EN 14214 (European Union) and ASTM D 6751 (United States) (Table 5). Depending on the catalyst, the ester content ranged between 99.1% and 99.9%. These values are significantly higher than the corresponding specied minimum value in the European Union standard (96.5%). Moreover, the amounts of all by-products analysed (glycerides, free glycerol, total glycerol and free fatty acids) were below the maximum values allowed by American and European standards. Besides, non-saponiable lipids were not detected in the M. circinelloides-derived biodiesel.

Table 5 Quality control of the biodiesel obtained in the direct transformation of M. circinelloides biomass. Property Catalyst BF3 Ester content (wt%) Monoglyceride content (wt%) Diglyceride content (wt%) Triglyceride content (wt%) Free glycerol (wt%) Total glycerol (wt%) Acid value (mg KOH/g) Non-saponiable lipids (wt%) Polar lipids (wt%)
a

EU Standard EN 14214 H2 SO4 99.9 n.da n.da 0.1 0.0030 0.0030 n.da n.da n.da HCl 99.5 n.da n.da n.da 0.0030 0.0030 n.da n.da 0.5 96.5 min. 0.8 max. 0.2 max. 0.2 max. 0.02 max. 0.25 max. 0.5 max. n.sa n.sa

U.S. Standard ASTM D6751

99.1 n.da n.da 0.8 0.0025 0.0025 n.da n.da 0.1

n.sa n.sa n.sa n.sa 0.02 max. 0.24 max. 0.5 max. n.sa n.sa

n.d = not detected; n.s = not specied limit.

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Therefore, these types of lipid were also eliminated during the purication stage, which is in contrast to the results obtained in the production of biodiesel with the previous extraction of lipids stage. Nonetheless, the biodiesel obtained had small amounts of polar lipids, which were lower than 0.5% in all cases (Table 5). Anyway, all the parameters analysed in the biodiesel produced in the one-step process developed here from the M. circinelloides biomass met both American and European standards, indicating that both storage lipids (glycerides and free fatty acids) and structural lipids (phospholipids, sphingolipids, saccharolipids, and sterol esters) were transformed into FAMEs. 4. Conclusions The present work shows that lipids from M. circinelloides may be a suitable feedstock for biodiesel production. The chloroform:methanol extraction system showed the highest lipid yield among the three extraction processes studied. FAMEs were produced by acid-catalysed transesterication and esterication at 65 C for 8 h comparing two different approaches: the transformation of previously extracted lipids and direct transformation from fungal biomass. This study shows that the direct method not only produces higher FAME purity (>99%) with all three catalysts employed (BF3 , H2 SO4 and HCl) but also, in addition, it increases FAME yields as a consequence of an improvement in lipid extraction and transformation. The characteristics of the M. circinelloides biodiesel suggest that biodiesel production by direct transformation of fungal biomass without an intermediate lipid extraction step is technically feasible, which represents a starting point for further studies aimed at assessing the development of this process on an industrial scale. Acknowledgements This work was funded by the D.G. de Investigacin y Poltica Cientca (Comunidad Autnoma de la Regin de Murcia, Spain), project BIO-BMC 07/01-0005. References
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