Bioresource Technology 153 (2014) 230–235

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Selection of oleaginous yeasts with high lipid productivity for practical biodiesel production Ayumi Tanimura a, Masako Takashima b, Takashi Sugita c, Rikiya Endoh b, Minako Kikukawa a, Shino Yamaguchi a, Eiji Sakuradani d, Jun Ogawa d, Jun Shima a,⇑ a

Research Division of Microbial Sciences, Kyoto University, Kitashirakawa Oiwake-Cho, Sakyo-ku, Kyoto 606-8502, Japan Japan Collection of Microorganisms, RIKEN BioResource Center, Koyadai, Tuskuba, Ibaraki 305-0074, Japan Department of Microbiology, Meiji Pharmaceutical University, Tokyo 204-8588, Japan d Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-Cho, Sakyo-ku, Kyoto 606-8502, Japan b c

h i g h l i g h t s  The lipid-producing ability of 500 newly isolated yeast strains was evaluated.  Lipid content, fatty acid composition, lipid productivity, and biomass were examined.  The yeast Cryptococcus musci was capable of the highest lipid productivity.

a r t i c l e

i n f o

Article history: Received 24 September 2013 Received in revised form 25 November 2013 Accepted 30 November 2013 Available online 8 December 2013 Keywords: Oleaginous yeast Fatty acids Lipid productivity Cryptococcus musci Cryptococcus podzolicus

a b s t r a c t The lipid-accumulating ability of 500 yeast strains isolated in Japan was evaluated. Primary screening revealed that 31 strains were identified as potential lipid producers, from which 12 strains were cultivated in a medium containing 3% glucose. It was found that JCM 24511 accumulated the highest lipid content, up to 61.53%, while JCM 24512 grew the fastest. They were tentatively identified as Cryptococcus sp. and Cryptococcus musci, respectively. The maximum lipid concentration of 1.49 g/L was achieved by JCM 24512. Similarly, JCM 24511 also achieved a high lipid production of 1.37 g/L. High lipid productivity is the most important characteristic of oleaginous yeasts from the viewpoint of practical production. Among the strains tested here, JCM 24512 had the best lipid productivity, 0.37 g/L/day. The results show that the isolated yeasts could be promising candidates for biodiesel production. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Biolipids, including triacylglycerol produced by oleaginous yeast, have been confirmed to be one of the most important raw materials for biodiesel production (Meng et al., 2009). The quality of biodiesel depends upon the fatty acid composition of the biolipids (Knothe, 2011). In general, biolipids produced by oleaginous yeast are suitable feedstock for biodiesel, because the fatty acid composition satisfies important criteria i.e., chain length and saturation degree. However, the fatty acid composition of biolipids is strain specific, it is therefore important to select oleaginous yeast strains to ascertain their suitability for biodiesel production. The other advantage of oleaginous yeast is their ability to produce lipids from non-utilized biomass, including lignocellulosic biomass (Gong et al., 2013; Liang et al., 2012). It is known that ⇑ Corresponding author. Tel.: +81 75 753 9545; fax: +81 75 753 9544. E-mail address: [email protected] (J. Shima). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.11.086

many oleaginous yeasts such as Rhodosporidium toruloides, Cryptococcus curvatus, Lipomyces starkeyi and Yarrowia lipolytica accumulate lipids to more than 20% of the dry yeast cells (Feofilova et al., 2010; Papanikolaou and Aggelis, 2010; Thiru et al., 2011; Wild et al., 2010; Wu et al., 2011) and that several oleaginous yeast, including R. toruloides and L. starkeyi, can assimilate xylose (Gong et al., 2012; Oguri et al., 2012; Zhao et al., 2012). One of the difficulties in the commercialization of biolipid production by oleaginous yeasts may be due to low productivity (Feofilova et al., 2010). In general, the growth of oleaginous yeasts is much slower, compared with that of ascomycetes such as Saccharomyces cerevisiae, and biolipid production by oleaginous yeasts requires a prolonged period to produce maximum yields (Ageitos et al., 2011). To evaluate the lipid-accumulating ability of oleaginous microorganisms, lipid content (% of dry cell weight) is the most commonly used parameter (Meng et al., 2009; Song et al., 2013). There are other derived values, for example, lipid concentration

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(g-lipid/L), lipid coefficient (lipid yield on glucose consumption; g-lipid/g-glucose), biomass concentration (g-cell/L) and lipid productivity (specific rate of lipid production; g-lipid/L/h or g-lipid/L/ day). However, lipid productivity is currently receiving a lot of attention as the selection of rapid-growing and accumulative strains is fundamental to the success of practical biodiesel production (Griffiths and Harrison, 2009; Ordog et al., 2013). Based on this background, the selection of oleaginous yeasts which can rapidly accumulate lipid, that is, high lipid productivity with a suitable fatty acid composition in typical culture media was undertaken. Recently, Takashima et al. (2012) reported the taxonomic richness of yeasts in Japan within subtropical and cool temperature areas. It can be considered that the yeast strains collected in Japan also exhibit functional diversity, including lipid-producing ability. Therefore, a comprehensive evaluation of the lipid-accumulating ability of yeast strains in the collection was performed. In this study, it was found that several yeast strains classified as basidiomycete, including Cryptococcus sp. and unclassified strains, accumulated biolipids with high productivity, compared with previously reported oleaginous yeasts. 2. Methods 2.1. Yeast strains Yeast strains collected and taxonomically identified by Takashima et al. were used as the main screening resource (Takashima et al., 2012). The yeast strains isolated from the campus of Kyoto University (Kyoto, Japan) were also assessed. As control strains, L. starkeyi NBRC 10381 and R. toruloides NBRC 0559 were obtained from the National Institute of Technology and Evaluation (NITE) Biological Resource Center. 2.2. Media YM agar medium (Difco, Detroit, MI, USA) was used for maintenance of yeast strains. YPD medium (1% yeast extract [Difco], 2% peptone [Difco] and 2% glucose) was used for the liquid cultivation of yeast cells, unless otherwise indicated. Synthetic defined (SD) medium (0.17% yeast nitrogen w/o ammonium sulphate and amino acids [Difco], 0.5% ammonium sulphate and 3% glucose) was used for the primary comprehensive screening of biolipidaccumulating ability. SS2 medium (3% glucose, 0.5% ammonium sulphate, 0.05% magnesium sulphate, 0.01% sodium chloride, 0.01% calcium chloride and 0.01% yeast extract [Difco]) was used for the secondary screening of oleaginous yeasts with high lipid productivity. 2.3. Measurement of intracellular fatty acids Total intracellular lipids were estimated as total fatty acids. The fatty acids of the yeast strains were extracted from the lyophilized cells using a hydrochloric acid-catalysed direct methylation (Ichihara and Fukubayashi, 2010). In brief, after cultivation, the yeast cells were harvested by centrifugation and lyophilization. The lyophilized cells were dissolved in toluene and methanol, then directly transmethylated with 8% methanolic HCl at 45 °C overnight. The resultant fatty acid methyl esters (FAMEs) were extracted with n-hexane and analysed using gas chromatography (GC-2010 Plus; Shimadzu, Kyoto, Japan) equipped with a flame ionization detector (FID) and an autosampler (AOC20; Shimadzu). A DB-23 capillary column (30 m  0.25 mm ID and 0.25 lm film thickness) (Agilent Technologies, Palo Alto, California, USA) was used. The column temperature was programmed to start at 50 °C

231

for 2 min and then increased by 10 °C/min up to 180 °C where it remained for 5 min, it was then increased at a rate of 5 °C/min to 240 °C, which was held for 3 min. Helium was the carrier gas, which was pumped at 1.0 mL/min, and nitrogen was used as the make-up gas. The injector temperature was 250 °C, the detector temperature was 300 °C, with a split ratio of 50:1. The identification of major peaks was performed based on retention time using controls obtained from Sigma–Aldrich (Saint Louis, MO, USA). The fatty acid concentrations were determined using a standard curve generated by a series of external standards. 2.4. Comprehensive evaluation of biolipid-accumulating ability of strains in the yeast collection Yeast strains were inoculated into 3 mL of YPD medium in test tubes and incubated overnight at 30 °C, with reciprocal shaking at 150 opm (preculture). The preculture was then suspended in 6 mL of SD medium in a test tube, to a cell optical density at 600 nm (OD600) of 0.2, then cultured at 30 °C with reciprocal shaking for 3 days at 150 opm. Cells from 3 mL of culture broth were harvested by centrifugation (15,000 rpm for 10 min) and washed twice with distilled water. Intracellular total lipids were determined after lyophilizing the wet cells (primary screening). 2.5. Selection of oleaginous yeasts which accumulate intracellular lipids at a high level The yeast strains screened by the primary screening were cultivated in Erlenmeyer flasks, containing 100 mL of SS2 medium at 27 °C, on a rotary shaker at 150 rpm. The preculture and cell dosage were the same as described above. Cells from 3 mL of culture broth were harvested after 1, 2, 3, and 4 days of cultivation, by centrifugation (15,000 rpm for 10 min), and washed twice with distilled water. Intracellular total lipids, fatty acid composition and cell mass were determined after lyophilizing the wet cells (secondary screening). All experiments were performed in triplicate. 2.6. Calculation of biodiesel properties Based on the equations of Hoekman et al. (2012), the biodiesel properties, namely viscosity, specific gravity, cloud point, cetane number, iodine number and higher heating value (HHV), were estimated. Average unsaturation (AU) was calculated from the compositional profiles in Table 2 as

AU ¼

X

N  Ci

ð1Þ

where N is the number of carbon–carbon double bonds of unsaturated fatty acids and Ci is the concentration (mass fraction) of the component. Each property was calculated using Eqs. (2)–(7) (Hoekman et al., 2012).

Viscosity ¼ 0:6316AU þ 5:2065

ð2Þ

Specific gravity ¼ 0:0055AU þ 0:8726

ð3Þ

Cloud point ¼ 13:356AU þ 19:994

ð4Þ

Cetane number ¼ 6:6684AU þ 62:876

ð5Þ

Iodine number ¼ 74:373AU þ 12:71

ð6Þ

HHV ¼ 1:7601AU þ 38:534

ð7Þ

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A. Tanimura et al. / Bioresource Technology 153 (2014) 230–235

accumulated total lipids to more than 0.8 g/L of the medium volume. The yeast strains examined in this study are listed in Table 1. As expected, the yeast strains which accumulated more than 0.8 g/ L belonged to the basidiomycetes (for details, see below) and were used for further analysis.

(a)

3.2. Screening of oleaginous yeasts which can accumulate biolipids at a higher level

12

(b)

300

Number of strains

250 200 150 100 50 0 0

0.2

0.4

0.2

0.4

0.6

0.6

0.8

1.0

1.2

1.4

0.8 1.0 1.2 1.4 Lipid concentration [g/L]

1.6

1.6

Fig. 1. Screening flow scheme (a) and the distribution of lipid accumulating ability in terms of lipid concentration during primary screening (b).

3. Results and discussion 3.1. Comprehensive evaluation of the biolipid-accumulating ability of strains in the yeast collection The screening flow scheme is shown in Fig. 1a. Thirteen strains with high lipid productivity were obtained through primary and secondary screening. During primary screening, a comprehensive evaluation of the lipid-accumulating ability of yeast strains (a total of 500 strains) was carried out. In this experiment, SD medium was employed, as almost all strains could grow in this medium. The distribution of the lipid accumulation level is indicated in Fig. 1b. Approximately 6% of the tested strains (total 31 strains)

During secondary screening, the evaluation of the lipidaccumulating ability and fatty acid composition of yeast strains which showed a lipid accumulation of more than 0.8 g/L in the primary screening was performed. In this experiment, SS2 medium was employed. The control strains used were L. starkeyi NBRC 10381 and R. toruloides NBRC 0559, which are known as typical oleaginous yeasts. The lipid-accumulating ability of the yeast strains is expressed as lipid content after 4 days of cultivation (Fig. 2). From the viewpoint of lipid content, many of the yeasts showed higher lipid-producing ability under experimental conditions used in this study, compared with those of the control strains. In particular, JCM 24511 showed the highest lipid content at 61.53 ± 2.25%, which was higher than that of the literature data; 22–31% from 3% glucose (Gouri et al., 2012). The fatty acid composition of lipids, produced by the yeast strains after 4 days, was examined (Table 2) as it is known to influence the quality of biodiesel produced from biolipids. No huge differences in fatty acid composition were observed between the 12 strains and control strains. Although palmitoleic acid was high in L. starkeyi NBRC 10381, at around about 4.7%, this did not influence the properties of the oil, such as oxidative stability (Hoekman et al., 2012). In most strains, the major fatty acids were the C18 species; for example, oleic acid (18:1) ranged from 55.43% to 72.95%; stearic acid (18:0) from 6.11% to 25.50% and linoleic acid (18:2) from 3.43% to 27.44%. The percentages of fatty acids remained at a constant level during cultivation. A suitable fatty acid composition for biodiesel production is palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic (18:3) acids (Knothe, 2009). The 12 selected strains contained these fatty acids at a high ratio, ranging from 92.77% to 97.17%. It can be considered that the fatty acids produced by the selected strains were suitable for biodiesel production. The lipid productivity (specific rate of lipid production) and biomass concentration of selected and control strains after 4 days of culturing are shown in Table 3. Every selected strain showed higher values, compared with the control strains, for both parameters. In terms of the lipid coefficient (g of lipid produced per g of glucose consumed), JCM 24502 and JCM 24511 reached the highest values at 0.17 ± 0.07 and 0.15 ± 0.03, respectively (data not shown). It was observed that the higher the lipid coefficient, the higher the

Table 1 Characteristics of selected 12 oleaginous yeasts. JCM number

Species

Phylogenetically close to

Higher taxa

Accession number

Source

JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM JCM

Cryptococcus sp. Cryptococcus ramirezgomezianus Cryptococcus podzoricus Cryptococcus podzoricus Cryptococcus sp. Cryptococcus sp. Cryptococcus podzoricus Cryptococcus sp. Cryptococcus podzoricus Cryptococcus sp. Cryptococcus musci Rhodotorula sp.

Cryptococcus podzoricus

Tremellales, Agaricomycotina Trichosporonales, Agaricomycotina Tremellales, Agaricomycotina Tremellales, Agaricomycotina Tremellales, Agaricomycotina Tremellales, Agaricomycotina Tremellales, Agaricomycotina Tremellales, Agaricomycotina Tremellales, Agaricomycotina Tremellales, Agaricomycotina Trichosporonales, Agaricomycotina Cystobasidiales, Pucciniomycotina

AB726647 AB726969 AB727275 AB726926 AB726487 AB726649 AB727220 AB726496 AB726880 AB726652 AB727274 AB727268

Soil, Soil, Soil, Soil, Soil, Soil, Soil, Soil, Soil, Soil, Soil, Soil,

24502 24503 24504 24505 24506 24507 24508 24509 24510 24511 24512 24513

Cryptococcus podzoricus Cryptococcus podzoricus Cryptococcus podzoricus Cryptococcus podzoricus Occultifur externus

Iriomote island Rishiri island Rishiri island Rishiri island Iriomote island Iriomote island Rishiri island Iriomote island Rishiri island Iriomote island Rishiri island Rishiri island

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A. Tanimura et al. / Bioresource Technology 153 (2014) 230–235 Table 2 Fatty acid composition of 12 selected oleaginous yeast strains and 2 control strains after a 4-day culture. Data are mean ± standard deviation of three assays.

JCM 24502 JCM 24503 JCM 24504 JCM 24505 JCM 24506 JCM 24507 JCM 24508 JCM 24509 JCM 24510 JCM 24511 JCM 24512 JCM 24513 L.starkeyi NBRC 10381 R.toruloides NBRC 0559

C12:0 lauric

C14:0 myristic

C16:0 palmitic

C16:1 palmitoleic

C18:0 stearic

C18:1 oleic

C18:2 linoleic

C18:3 linolenic

C22:0 behenic

C24:0 lignoceric

0.01 ± 0.01 0.11 ± 0.01 ND ND ND ND ND ND ND ND 0.06 ± 0.01 0.03 ± 0.02 ND

0.38 ± 0.03 1.02 ± 0.10 0.37 ± 0.03 0.26 ± 0.02 0.40 ± 0.01 0.39 ± 0.02 0.36 ± 0.02 0.29 ± 0.02 0.15 ± 0.02 0.33 ± 0.03 0.82 ± 0.13 0.95 ± 0.10 0.61 ± 0.01

3.00 ± 0.13 2.92 ± 0.15 2.64 ± 0.06 2.39 ± 0.23 2.86 ± 0.04 2.99 ± 0.08 2.87 ± 0.06 2.63 ± 0.22 1.64 ± 0.13 2.75 ± 0.06 2.95 ± 0.05 3.51 ± 0.14 4.34 ± 0.14

0.13 ± 0.02 0.67 ± 0.06 0.14 ± 0.04 0.09 ± 0.02 0.09 ± 0.01 0.11 ± 0.01 0.14 ± 0.01 0.11 ± 0.08 0.05 ± 0.04 0.09 ± 0.01 0.22 ± 0.02 0.57 ± 0.07 4.70 ± 0.17

19.18 ± 0.43 9.74 ± 0.91 14.07 ± 1.48 16.52 ± 1.41 22.34 ± 1.06 21.48 ± 0.54 14.53 ± 0.17 25.50 ± 1.21 23.70 ± 0.43 21.21 ± 0.90 20.03 ± 0.99 6.11 ± 0.28 6.03 ± 0.43

69.02 ± 0.28 55.43 ± 1.80 72.95 ± 1.25 71.68 ± 1.42 66.29 ± 1.06 65.98 ± 0.57 70.48 ± 1.34 62.57 ± 0.60 62.84 ± 1.17 67.37 ± 1.25 63.09 ± 0.43 68.95 ± 0.35 74.08 ± 0.29

4.28 ± 0.23 27.44 ± 2.28 5.38 ± 0.31 4.06 ± 0.15 3.43 ± 0.30 5.29 ± 0.14 7.44 ± 0.98 4.24 ± 0.69 3.92 ± 0.40 3.91 ± 0.39 9.98 ± 1.41 18.56 ± 0.15 6.75 ± 0.60

0.45 ± 0.03 0.70 ± 0.11 0.82 ± 0.04 0.57 ± 0.01 0.38 ± 0.04 0.40 ± 0.01 0.74 ± 0.12 0.44 ± 0.09 0.67 ± 0.05 0.41 ± 0.05 0.31 ± 0.03 0.05 ± 0.04 0.71 ± 0.07

1.95 ± 0.01 0.46 ± 0.02 2.00 ± 0.13 1.84 ± 0.14 2.35 ± 0.08 2.09 ± 0.04 2.16 ± 0.14 2.04 ± 0.08 3.13 ± 0.16 2.13 ± 0.07 0.95 ± 0.07 0.24 ± 0.02 0.51 ± 0.05

1.61 ± 0.14 1.53 ± 0.06 1.71 ± 0.23 2.58 ± 0.31 1.86 ± 0.04 1.27 ± 0.08 1.58 ± 0.18 2.25 ± 0.38 3.94 ± 0.60 1.99 ± 0.09 1.71 ± 0.28 1.21 ± 0.09 2.27 ± 0.04

ND

1.31 ± 0.07

1.86 ± 0.10

0.43 ± 0.06

17.60 ± 0.53

64.21 ± 0.54

9.11 ± 1.24

1.64 ± 0.11

1.11 ± 0.02

2.74 ± 0.12

ND: not detected.

70

Lipid content [%, DW]

60 50 40 30 20

R. toruloides NBRC 0559

JCM 24513

L. starkeyi NBRC 10381

JCM 24512

JCM 24511

JCM 24510

JCM 24509

JCM 24508

JCM 24507

JCM 24506

JCM 24505

JCM 24504

JCM 24503

JCM 24502

0

JCM 24501

10

Fig. 2. Comparison of lipid content of 12 selected oleaginous yeast strains and 2 control strains after a 4-day culture. Data are mean ± standard deviation (error bars) of three assays.

Table 3 Lipid productivity (lipid concentration per day) and biomass concentration of 12 selected oleaginous yeast strains and 2 control strains after a 4-day culture. Data are mean ± standard deviation of three assays.

JCM 24502 JCM 24503 JCM 24504 JCM 24505 JCM 24506 JCM 24507 JCM 24508 JCM 24509 JCM 24510 JCM 24511 JCM 24512 JCM 24513 L. starkeyi NBRC 10381 R. toruloides NBRC 0559

Lipid productivity (g/L/day)

Biomass concentration (g/L)

0.35 ± 0.05 0.14 ± 0.02 0.21 ± 0.03 0.30 ± 0.11 0.29 ± 0.07 0.26 ± 0.04 0.31 ± 0.08 0.30 ± 0.02 0.16 ± 0.05 0.34 ± 0.04 0.37 ± 0.15 0.22 ± 0.03 0.14 ± 0.02 0.12 ± 0.01

2.44 ± 0.36 2.46 ± 0.05 1.98 ± 0.54 2.18 ± 0.64 2.00 ± 0.43 2.43 ± 0.30 2.52 ± 0.29 2.16 ± 0.12 1.59 ± 0.15 2.23 ± 0.29 3.27 ± 0.35 2.76 ± 0.48 1.39 ± 0.25 1.25 ± 0.06

efficiency of glucose utilization. Thus, these strains, phylogenetically close to Cryptococcus podzolicus, were efficient in converting glucose into lipid, which would enable the use of a production process of lower cost. On the other hand, JCM 24512 showed the highest biomass concentration of 3.27 ± 0.35 g/L and the highest lipid productivity of 0.37 ± 0.15 g/L/day, while its lipid content and lipid coefficient were not so high. These parameters are related to each other and it can be concluded that the lipid content of each cell of JCM 24512 was dispersed into small fat droplets, but lipid productivity was high due to the large cell mass. In scaling-up the fermentation process, the lipid amount per media volume per day correlates closely with production efficiency. Consequently, it is considered that the most important parameter is lipid productivity. High lipid productivity increases yield per harvest volume and decreases the production cost. Therefore, it was suggested that JCM 24512 could play a key role in the economic production of biodiesel. Similarly, JCM 24502 and JCM 24511 also achieved high lipid productivity, which were close to C. podzolicus. Surprisingly, among the 12 selected strains, 9 strains belong to or are close relatives of C. podzolicus (Takashima et al., 2012). This is the first report for the accumulation of lipids using Cryptococcus musci and C. podzolicus. Microalgae have been considered another candidate for lipid production. However, although microalgae accumulate lipids at high levels (60–70% of dry cell weight; Song et al., 2013), they require a long fermentation period of 15 days or more, a large culturing space and sufficient sunlight energy to convert carbon dioxide to lipids (Meng et al., 2009). In addition to the other requirements, the larger culturing space would increase the investment in the facilities and equipment. On the other hand, open-pond systems require carefully selected environments due to the threat of contamination and pollution from other microorganisms (Brennan and Owende, 2010). Moreover, the technique for extracting lipids from microalgae is still in the development stage (Pragya et al., 2013). In contrast, oleaginous yeasts can be cultivated in a tank with a capacity of a few hundred liters and do not require a photobioreactor. Considering the total cost and technologies for production, oleaginous yeasts are a potential alternative resource for biodiesel production. 3.3. Properties of the biodiesel from oleaginous yeasts The measurement of biodiesel properties requires a considerable amount of oil sample and a specialized apparatus. To

A. Tanimura et al. / Bioresource Technology 153 (2014) 230–235

3.4. Kinetic analysis of yeast strains with high lipid productivity To examine the lipid-accumulating rate of yeast strains with high lipid productivity, the kinetic analysis of three yeast strains (JCM 24502, JCM 24511 and JCM 24512) was performed, which were compared with the control strains L. starkeyi NBRC 10381 and R. toruloides NBRC 0559. The time course of lipid concentration

(a)

Lipid concentration [g/L]

overcome this limitation, predictive equations were recently developed and have generally proven successful in estimating the physical properties of oil composed of FAMEs (Felipe Ramirez-Verduzco et al., 2012; Hoekman et al., 2012; Knothe and Steidley, 2011). In the present study, the equations of Hoekman et al. (2012) were employed to predict the biodiesel properties, such as the viscosity, specific gravity, cloud point, cetane number, iodine number and HHV. Table 4 summarizes these values for 14 biolipids and plant oils (rapeseed oil and jatropha oil). The values in brackets are the measured properties (Hoekman et al., 2012) and were in reasonably good agreement with the calculated data, except in the case of the cloud point of rapeseed oil. Generally, the measured properties were correlated with the degree of unsaturation. The US and European specifications for biodiesel, ASTM 6751 and EN 14214, are also listed in Table 4 (Hoekman et al., 2012). The values of viscosity, specific gravity, cetane number and iodine number of biolipids from 12 selected oleaginous yeasts were close to the values of plant oils and satisfied the specifications. There is no cloud point value in either set of specifications due to the seasonal and geographic temperature variability in this parameter. However, compared to the plant oils, the biolipids from oleaginous yeasts showed relatively high cloud points. These higher cloud points are considered undesirable and were attributed to the low temperature properties of biodiesel. It has been reported that higher cloud points result from the presence of saturated methyl esters longer than C12 (Hoekman et al., 2012). It may concluded that the high content of C18:0 depresses the low temperature performance of biolipids; the mass fractions of C18:0 of rapeseed oil and jatropha oil were 1.6% and 6.1%, respectively (Hoekman et al., 2012). HHV is relevant to the energy content of the oil, and a higher value is preferable. The HHV values of biolipids from 12 selected oleaginous yeasts were around 40 and almost the same as those of plant oils.

(b)

2

1.5

1

0.5

0 3.5 0

1

2

1

2

3

4

3

4

3

Biomass concentration [g/L]

234

2.5 2 1.5 1 0.5 0 0

day Fig. 3. Time course of lipid concentration (a) and biomass concentration (b) of JCM 24502 (filled circle), JCM 24511 (open circle), JCM 24512 (asterisk), L. starkeyi (filled triangle) and R. toruloides (open triangle). Data are mean ± standard deviation (error bars) of three assays.

and biomass concentration of the three yeast strains and control strains are shown in Fig. 3. JCM 24512 gave the highest rate of increase of lipid concentration and biomass. It should be noted that

Table 4 Comparisons of biodiesel properties from oleaginous yeasts with rapeseed oil and jatropha oil and the US biodiesel and EU biodiesel standards.

JCM 24502 JCM 24503 JCM 24504 JCM 24505 JCM 24506 JCM 24507 JCM 24508 JCM 24509 JCM 24510 JCM 24511 JCM 24512 JCM 24513 L. starkeyi NBRC 10381 R. toruloides NBRC 0559 Rapeseed oil Jatropha oil US biodiesel standard ASTM D6751 EU biodiesel standard EN 14214

Viscosity (mm/s2)

Specific gravity

Cloud point (°C)

Cetane number

Iodine number

HHV (MJ/kg)

4.71 4.49 4.66 4.69 4.74 4.71 4.65 4.75 4.75 4.72 4.67 4.53 4.61 4.65 4.40 (4.50) 4.48 (4.75) 1.9–6.0 3.5–5.0

0.8769 0.8788 0.8773 0.8771 0.8767 0.8769 0.8774 0.8766 0.8766 0.8768 0.8772 0.8785 0.8778 0.8774 0.8796 (0.879) 0.8789 (0.876) – 0.86–0.9

9.43 4.89 8.47 9.10 10.06 9.59 8.28 10.31 10.28 9.78 8.75 5.73 7.38 8.27 2.93 (-3) 4.67 (5) – –

57.60 55.34 57.12 57.43 57.92 57.68 57.03 58.04 58.03 57.77 57.26 55.75 56.58 57.02 54.35 (53.7) 55.23 (55.7) 47 min 51 min

71.51 96.81 76.90 73.40 68.03 70.62 77.95 66.62 66.81 69.61 75.33 92.13 82.93 77.99 107.76 (116.1) 98.02 (109.5) – 120 max

39.93 40.52 40.05 39.97 39.84 39.90 40.08 39.81 39.81 39.88 40.02 40.41 40.20 40.08 40.78 (41.1) 40.55 (40.7) – –

The values in brackets are from a previous report (Hoekman et al., 2012).

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the lipid concentration of JCM 24512 reached the maximum value of 1.57 ± 0.38 g/L at 3 day, and then slightly decreased, while the biomass kept increasing until 4 day. This may suggest that it was related to factors influencing the exhaustion of essential elements. As can be seen, however, the lipid productivity of the first two days of JCM 24512 was remarkable (0.66 ± 0.14 g/L/day). This value was higher than that of published data; approx. 0.5 g/L/day from 3% glucose (Gouri et al., 2012). The result confirmed that JCM 24512 can accumulate lipid rapidly along with high yield. This strain can potentially shorten fermentation times and reduce production costs. Generally, fermentation with oleaginous yeast requires extensive time to reach the required lipid yield (Ageitos et al., 2011; Beopoulos et al., 2009), as lipid accumulation in oleaginous yeast is known to occur during stress conditions in the medium (Ratledge and Wynn, 2002). When cells begin the depletion of nutrients, such as nitrogen, the excess carbon in the culture medium is converted into cellular lipid (Ageitos et al., 2011). This pattern is observed in oleaginous yeast and filamentous fungi (Ratledge and Wynn, 2002). The lipid-accumulating behaviour of JCM 24512 differed from the other 4 strains, suggesting that the lipid-accumulating mechanism of JCM 24512 was less related to the nitrogen concentration. Further investigation is needed to clarify the effect of nitrogen concentration on the lipid-accumulating pattern of this strain. 4. Conclusion The results indicate that the yeast strains isolated from Japan have great potential for biodiesel production. JCM 24502 and JCM 24511, tentatively identified as Cryptococcus sp., reached a lipid content of 56.77 ± 2.80% and 61.53 ± 2.25%, respectively. Based on these studies it was concluded that the most promising strain was Cryptococcus musci JCM 24512, with a lipid content of 44.7 ± 15.04% and lipid productivity of 0.37 ± 0.15 g/L/day; during the first 2 days of cultivation this reached 0.66 ± 0.14 g/L/day. High lipid productivity is a crucial characteristic of any species used for biodiesel production. Acknowledgements This work was partly supported by the Institute for Fermentation, Osaka (IFO) and Advanced Low Carbon Technology Research and Development Program of Japan (ALCA). References Ageitos, J.M., Vallejo, J.A., Veiga-Crespo, P., Villa, T.G., 2011. Oily yeasts as oleaginous cell factories. Appl. Microbiol. Biotechnol. 90, 1219–1227. Beopoulos, A., Cescut, J., Haddouche, R., Uribelarrea, J.-L., Molina-Jouve, C., Nicaud, J.-M., 2009. Yarrowia lipolytica as a model for bio-oil production. Prog. Lipid Res. 48, 375–387. Brennan, L., Owende, P., 2010. Biofuels from microalgae–a review of technologies for production, processing, and extractions of biofuels and co-products. Renew. Sust. Energ. Rev. 14, 557–577.

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