Screening high oleaginous Chlorella strains from different climate zones

Bioresource Technology 144 (2013) 637–643

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Screening high oleaginous Chlorella strains from different climate zones Jin Xu, Hanhua Hu ⇑ Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China

h i g h l i g h t s  23 Oil-producing Chlorella strains from different climate zones were obtained.  Four Chlorella strains with high triacylglycerol content were cultured at 5–40 °C.  Chlorella sp. NJ-18 displayed higher triacylglycerol productivity at 5–30 °C.  Accumulation of triacylglycerols in strain NMX35N changed a little from 30 to 40 °C.  NJ-18 and NMX35N are promising as feedstock of biodiesel at various temperatures.

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Article history: Received 20 May 2013 Received in revised form 6 July 2013 Accepted 8 July 2013 Available online 13 July 2013 Keywords: Chlorella sp. Temperature Fatty acids Triacylglycerol Biodiesel

a b s t r a c t In outdoor cultivation, screening strains adapted to a wide temperature range or suitable strains for different environmental temperatures is of great importance. In this study, triacylglycerol (TAG) content of 23 oil-producing Chlorella strains from different climate zones were analyzed by thin layer chromatography. Four selected Chlorella strains (NJ-18, NJ-7, NMX35N and NMX139N) with rather high TAG content had higher total lipid content compared with Chlorella vulgaris SAG 211-11b. In particular, NJ-18 displayed the highest TAG productivity among the four high oil-producing Chlorella strains. Accumulation of TAGs in strain NMX35N changed a little from 30 to 40 °C, showing a desirable characteristic of accumulating TAGs at high temperatures. Our results demonstrated that NJ-18 and NMX35N had suitable fatty acid profiles and good adaption to low and high temperatures respectively. Therefore, cultivation of the two Chlorella strains together might be a good option for economic biodiesel production during the whole seasons of the year. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Since fossil fuel is not sustainable and its usage may cause serious environmental problems, more and more attention has been attached to clean renewable energy (Borowitzka and Moheimani, 2013; Chisti, 2008; Hu et al., 2008b; Huang et al., 2010; Jones and Mayfield, 2012). Biodiesel (fatty acid methyl esters), derived from triacylglycerols (TAGs) by transesterification with methanol, is a renewable, biodegradable, and nontoxic fuel (Chisti, 2007). Currently, it is produced mainly from vegetable oils and animal fats, however, the large-scale production of biodiesel from them is unsustainable and uncompetitive in cost (Chisti, 2008). Microalgae as feedstock for biodiesel can serve as an alternative and potential candidate, based on the high photosynthetic efficiency and lipid productivity, and their ability to grow rapidly, tolerate and adapt to a variety of environmental conditions (Chisti, 2007; Hu et al., 2008b).

⇑ Corresponding author. Tel./fax: +86 27 68780078. E-mail address: [email protected] (H. Hu). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.07.029

Screening an optimal oleaginous microalga for outdoor cultivation is the very first and most important step toward the utilization of microalgae as feedstock for biodiesel production (Scott et al., 2010). Temperature is an important factor for the outdoor cultures, and it has a great influence on the growth and lipid content of microalgae (Li et al., 2013). Therefore, the ability of microalgae to acclimate to variable outdoor temperatures should be taken into account in the screening process. Chlorella strains are regarded as competent candidates for biodiesel production due to their fast growth and easier cultivation (Mata et al., 2010). They distribute all over the world including polar areas (Ahn et al., 2012; Hoek et al., 1995; Hu et al., 2008a), showing an extensive adaptability to changing temperatures (2–42 °C) (Ahn et al., 2012; Hu et al., 2008a; Huss et al., 1999; Kessler, 1985), and the optimal growth temperature and the maximum lipid productivity are strain-specific. For example, Chlorella vulgaris tended to stop growing at temperature above 30 °C (Kessler, 1985), while Chlorella sorokiniana exhibited a better growth with an increase of temperature from 28 to 42 °C (de-Bashan et al., 2008). In addition, lipid content and fatty acid composition are influenced greatly by temperature (Converti et al., 2009; Li et al.,

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2013). Therefore, to reduce the cultivation expenditure it is very important to screen strains with a capacity of adapting to a wide range of temperature or find suitable strains for different environmental temperatures. In this study, Chlorella strains isolated from Antarctica and different climate zones in China were screened by thin layer chromatography (TLC) for a rough identification of high lipid productive stains on the basis of the TAG content. Selected high oleaginous stains were grown under different cultivation temperatures and evaluated by Nile Red dye to define those with high lipid productivity, hence obtaining Chlorella strains that can serve as feedstock for biodiesel production at different seasons.

2.3. Preliminary screening of oleaginous strains All Chlorella strains were grown in 50 ml flasks containing 20 ml BG11 liquid medium (the starting optical density OD750 = 0.05) at 25 °C under continuous illumination of 100 lmol photons m2 s1 for 20 d, then total lipid was extracted from the cell pellet and analyzed using TLC for the comparison of the TAG content. The diatom Phaeodactylum triconutum with high TAG content was used as control. TLC was performed as described by Reiser and Somerville (1997) by one-dimensional TLC on silica gel plates 60 F254 (Merck KgaA, Darmstadt, Germany). Standard triolein was purchased from Sigma–Aldrich Corporation (St. Louis, MO, USA).

2. Methods 2.4. Growth of high oil-producing Chlorella at different temperatures 2.1. Strains Water samples from subtropical (Hubei Province) and warm temperate zone (Jiangsu Province and Inner Mongolia Autonomous Region) in China were collected and plated on BG11 medium (Stanier et al., 1971) solidified with 1.5 % agar. Single green algal colonies were obtained and further purified by repeated streaking on solid BG11 medium. The unialgal cultures of genus Chlorella were used in this study. C. vulgaris NJ-7 and Chlorella sp. NJ-18 were isolated from the Zhongshan Station of Antarctica by our lab in January 1999 (Hu et al., 2008a). C. vulgaris SAG 211-11b was purchased from the Culture Collection of Algae at the University of Texas (Austin, TX, USA). FACHB3, FACHB31, FACHB270 and FACHB275 were obtained from the FACHB-Collection (Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China). 2.2. Phylogenetic analysis The total genomic DNA was extracted from algal cells using a glass milk DNA isolation kit (Fermentas, Vilnius, Lithuania) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) was performed by using the general primers 18S-1 (50 -tggttgatcctgccagtagtc-30 ) and 18S-2 (50 -tgatccttctgcaggttcacc30 ) to amplify 18S rDNA gene as previously described (Hu et al., 2008a). The PCR products were excised from agarose gel and recovered with the DNA isolation kit, cloned into pMD18-T (Takara, Dalian, China) and sequenced. All ‘‘true’’ Chlorella species (Huss et al., 1999; Krienitzi et al., 2004) and some oil-producing Chlorella species sensu lato were included in phylogenetic analysis. Phylogenetic analyses were performed by PAUP4.0b (Swofford, 1998), with 1000 bootstrap replicates for neighbor-joining and parsimony analyses, 100 replicates for the maximum likelihood analysis using Gloeotilopsis planctonica (Z28970) and Ulothrix zonata (Z47999) as the outgroup.

Four high oil-producing Chlorella stains (C. vulgaris NJ-7, Chlorella sp. NJ-18, Chlorella sp. NMX35N and Chlorella sp. NMX139N) determined by preliminary screening and the type species of Chlorella, C. vulgaris SAG 211-11b, were grown in 250 mL flasks containing 150 mL BG11 (NaNO3 concentration was reduced to 880 lM) liquid medium (the starting optical density OD750 = 0.05) at 5, 15, 20, 25, 30, 35 and 40 °C under continuous illumination of 100 lmol photons m2 s1. Each was performed in triplicate. Cell density was determined by measuring OD750 every 2 days and specific growth rates (l) were calculated with the formula l = (lnXt  lnX0)/t, in which X0 is the initial cell density and Xt is the cell density after t days. 2.5. Total lipid extraction and fatty acid analysis Four high oil-producing Chlorella stains and C. vulgaris SAG 21111b were grown in 2 L flasks containing 1.5 L BG11 liquid medium bubbled with aseptic air at 25 °C under continuous illumination of 150 lmol photons m2 s1. Cells were harvested during the stationary phase by centrifugation (6000g, 5 min) and washed for three times with deionized water, then freeze-dried. Total lipids of each sample were extracted from approximately 100 mg dry cells and lipid contents (dry weight) were measured as described by Bligh and Dyer (1959). TAGs were separated and quantified by one-dimensional TLC on silica gel plates 60 F254 (Merck KgaA, Darmstadt, Germany), and the bands were identified by staining with iodine and then scraped off the plates. TAGs fractionated from the TLC plate and total lipids were methylated according to Hu et al., (2008a). Fatty acid methyl esters were identified and quantified by gas chromatography (TRACE GC, Thermo Scientific, Milan, Italy) equipped with a split/splitless injector, a flame ionization detector (FID) and a capillary column (60 m  0.25 mm) (DB-23, J&W Scientific, USA). Two microliters of each sample were injected in the splitless injection mode. The

Fig. 1. Analysis of the total lipids extracted from microalgal cultures by thin layer chromatography (TLC). The diatom P. triconutum with high TAG content was used as control. 1–23: Chlorella strains (1: NXM2, 2: NXM44, 3: NMX139N, 4: THX130, 5: THX193, 6: THX129, 7: FACHB275, 8: HBX169, 9: FACHB31, 10: THX132, 11: HBX356, 12: NMX35N, 13: NMX310, 14: HBX326, 15: HBX402, 16: YEL, 17: NJ-7, 18: NMX1, 19: NJ-18, 20: FACHB270, 21: FACHB3, 22: NMX37N, 23: NMX36); M: triolein (0.02 mg); C: P. triconutum; T: C. vulgaris SAG 211-11b. TAGs were indicated by arrows.

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Gloeotilopsis planctonica SAG 29.93 (Z28970) Ulothrix zonata SAG 38.86 (Z47999) Chlorella zofingiensis SAG 211-14a (X74004) 100/99/100 Graesiella emersonii NIES-2151 (AB488562) Chlorella mirabilis Andreyeva 748-I (X74000) 100/99/100 Chlorella ellipsoidea SAG 211-1a (X63520) Chlorella minutissima C-1.1.9 (X56102) Auxenochlorella protothecoides SAG 211-7a (X56101) Parachlorella kessleri SAG 211-11g (X56105) Chlorella sp. NJ-18 (DQ377324) Chlorella sp. NDem 9/21 T-13d (AY197628) 97/90/100 Chlorella sp. NMX37N Chlorella sp. NMX1 Chlorella lobophora Andreyeva 750-I (X63504) Chlorella sp. Mary 9/21 BT-10w (AY197620) 61/52/60 Chlorella sp. HBX326 Chlorella sp. NMX44 Chlorella sp. YEL (DQ377322) Chlorella sp. IFRPD 1018 (AB260898) Chlorella sp. HBX402 Chlorella sorokiniana SAG 211-8k (X62441) 97/75/65 Chlorella sp. THX132 Chlorella vulgaris IAM C-27 (AJ242757) 96/94/100 Chlorella vulgaris SAG 211-11b (X13688)/NJ-7 Chlorella sp. THX130 Chlorella sorokiniana CCALA 260 (FM205860) Chlorella sp. FACHB270 Chlorella sp. NMX35N Chlorella sp. NMX2 Chlorella sp. THX129 Chlorella sp.HBX169 Chlorella sp. FACHB275 Chlorella sp. HBX356 Chlorella sorokiniana Prag A14 (X74001) Chlorella sp. FACHB3 Chlorella sp. FACHB31 Chlorella pyrenoidosa SUN CHLORELLA (AB240151) Chlorella sp. MBIC10595 (AB058372) 85/81/72 Chlorella sp. NMX310 Chlorella sp. NMX139N Chlorella sp. THX193 65/66/71 Chlorella sp. VI2 (FJ946883) Hegewaldia sp. NMX36 99/99/100 Hegewaldia parvula CCAP 283/2 (FM205843) Chlorella saccharophila SAG 211-9a (X63505) 100/99/100 Heterochlorella luteoviridis SAG 211-2a (X73997) 100/99/100

True Chlorella

50

Fig. 2. Maximum-likelihood tree of Chlorella species sensu lato inferred from 18S rRNA gene sequences (strains obtained during this study are underlined). Bootstrap values are shown at the internal nodes for maximum likelihood (ML; 100 replications), neighbor joining (NJ; 1000 replications), and maximum parsimony (MP; 1000 replications), respectively, if the node is supported by at least two bootstrap values of 50% or above. Branch lengths correspond to evolutionary distances. A distance of 50 is indicated by the scale.

injector and detector were set at 270 °C and the column was programmed as follows: initial temperature 50 °C, isothermal for 1 min, then 40 °C min1 up to 170 °C, isothermal for 1 min, then 18 °C min1 up to 210 °C, and isothermal for 28 min. Nitrogen gas was used as the carrier gas. Fatty acids were identified by comparison of their retention times with those of standards (Sigma). Heptadecanoic acid (C17:0) was used as internal standard. 2.6. Neutral lipid accumulation during the batch culture at different temperatures C. vulgaris SAG 211-11b and three high oil-producing Chlorella stains (C. vulgaris NJ-7, Chlorella sp. NJ-18, and Chlorella sp.

NMX35N) with different ranges of growth temperature were grown in 250 mL flasks containing 150 mL BG11 (NaNO3 concentration was reduced to 880 lM) liquid medium (the starting optical density OD750 = 0.05) at 5, 15, 20, 25, 30, 35 and 40 °C under continuous illumination of 100 lmol photons m2 s1, and each was performed in triplicate. Sampling was performed every two days to determine cell density, nitrate concentration, and neutral lipid content. Cell density was determined by measuring OD750. Nitrate concentration in BG11 medium was evaluated spectrophotometrically at 220 nm according to the method described by Collos et al., (1999). The relative abundance of intracellular neutral lipid present in samples from Chlorella cultures was estimated by fluorometric assay using

Table 1 The upper and lower limits of growth temperature in some common Chlorella species sensu lato. Species

Lower (°C)

Upper (°C)

References

Chlorella sorokinina Chlorella vulgaris Chlorella lobophora Chlorella pyrenoidosa Chlorella sp. NJ-18 Chlorella sp. NMX37N Chlorella sp. YEL Chlorella sp. FACHB31 Chlorella sp. NMX35N Chlorella sp. NMX139N Auxenochlorella protothecoides ‘‘Chlorella’’ zofingiensis ‘‘Chlorella’’ minutissima Parachlorella kessleri

14 2–5 – 15 4 15 15 15 15 15 – – – –

36–42 28–32 30 40–43 30 35 40 40 40 40 28–34 28 32 34–36

Huss et al., (1999); Kessler (1985) ; Patterson, (1970) Huss et al. (1999); Kessler (1985) Huss et al. (1999) Sorokin and Krauss (1962) Hu et al. (2008a); This study Xu et al. (2012) Hu et al. (2008a) Hu et al. (2008a) This study This study Huss et al. (1999); Kessler (1985) Huss et al. (1999); Kessler (1985) Huss et al. (1999); Kessler (1985) Huss et al. (1999); Kessler (1985)

3.51 ± 0.16 0.12 ± 0.08 31.18 ± 1.44 1.4 ± 0.03 2.54 ± 0.2 5.68 ± 0.12 49.17 ± 1.62 0.11 ± 0.06 6.17 ± 0.36 0.11 ± 0.01 29.77 ± 0.85

0.2

Total lipids

0.4

NMX139N

µ (d -1)

0.6

4.29 ± 0.07 0.04 ± 0 33.87 ± 0.66 1.59 ± 0.02 2.38 ± 0.14 6.52 ± 0.44 45.66 ± 0.86 0.03 ± 0.01 5.2 ± 0.49 0.27 ± 0.11 12.99 ± 1.06

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Triacylglycerol

640

Chlorella vulgaris NJ-7

Triacylglycerol

Chlorella vulgaris SAG 211-11b Chlorella sp. NMX35N Chlorella sp. NMX139N

0.0 5

10

15

20

25

30

35

40

Temperature (°C)

0.72 ± 0.03 0.12 ± 0.01 33.3 ± 0.58 0.25 ± 0.01 3.85 ± 0.03 15.77 ± 0.01 36.34 ± 0.4 0.13 ± 0.01 9.51 ± 0.21 0.01 ± 0.01 12.18 ± 1.15

Chlorella sp. NJ-18

0.51 ± 0.04 0.09 ± 0 30.02 ± 1.22 1.98 ± 0.71 4.16 ± 0.22 16.93 ± 0.12 37.01 ± 0.37 0.13 ± 0.02 9.15 ± 0.52 0.03 ± 0 24.81 ± 1.15

Total lipids Triacylglycerol

0.33 ± 0.01 0.02 ± 0.01 22.11 ± 0.5 0.81 ± 0.01 1.58 ± 0.01 45.26 ± 0.11 8.81 ± 0.05 0.1 ± 0 20.97 ± 0.53 Not detected 6.11 ± 0.96 0.61 ± 0.05 0.05 ± 0.05 25.42 ± 0.03 0.73 ± 0.07 1.88 ± 0.3 45.55 ± 0.05 7.89 ± 0.24 0.13 ± 0.03 17.72 ± 0.11 0.02 ± 0 20.26 ± 0.91

Total lipids Triacylglycerol

1.09 ± 0.06 0.030 ± 0 23.99 ± 1.76 1.06 ± 0.06 1.76 ± 0.04 31.56 ± 0.87 32.8 ± 0.79 0.05 ± 0.01 7.66 ± 0.28 Not detected 20.22 ± 0.16 1.0 ± 0.11 0.32 ± 0.39 22.81 ± 1.74 0.87 ± 0.01 1.89 ± 0.27 33.92 ± 1.59 32.45 ± 2.25 0.04 ± 0.01 6.69 ± 1.05 Not detected 22.46 ± 1.24

Total lipids

0.49 ± 0.03 0.15 ± 0.01 20.96 ± 0.07 0.92 ± 0.01 0.69 ± 0.04 34.0 ± 1.6 16.72 ± 0.53 0.14 ± 0.02 25.9 ± 1.1 0.021 ± 0 20.46 ± 0.56

Triacylglycerol Total lipids

0.66 ± 0.15 0.14 ± 0.01 23.91 ± 0.96 0.84 ± 0.03 0.76 ± 0.06 35.14 ± 0.31 15.9 ± 0 0.09 ± 0 22.16 ± 0.02 0.31 ± 0.44 31.71 ± 1.42 C14:0 C14:1 C16:0 C16:1 C18:0 C18:1 C18:2 c-C18:3 a-C18:3 C20:0 % Of dcw

NMX35N SAG 211-11b

Microalgae are an extremely diverse group that contains many thousands of known species, and potentially hundreds of thousands. The great diversity provides a wide range of starting strains for fuel production. The extant green algae consist of approximately 8000 species and can be found in various environments including soil, humid rock, freshwater, and ocean (Hoek et al., 1995). Among them, Chlorella has been identified as a good option for biodiesel production (Mata et al., 2010). In this study, a total of 16 oil-producing freshwater Chlorella species were isolated, including 7 from Inner Mongolia (NMX1, NMX2, NMX44, NMX310, NMX35N, NMX37N, and NMX139N), 5 from Hubei Province (HBX169, HBX326, HBX356, HBX402, and YEL), and 4 from Jiangsu Province (TH129, THX130, THX132, and THX193) in China. In addition, 2 Antarctic Chlorella strains (NJ-7 and NJ-18) isolated by our lab (Hu et al., 2008a), 4 oil-producing Chlorella strains obtained from FACHB (FACHB3, FACHB31, FACHB270, and FACHB275), and the type strain of this genus C. vulgaris SAG 211-11b were also studied. Since biodiesel is produced by the transesterification of TAG with methanol (or other alcohol), high TAG content is one of the desirable features for using microalgae as biodiesel feedstock. To evaluate the TAG content, the total lipids extracted from the Chlorella cultures were analyzed by the TLC (Fig. 1). TAG contents in these strains were different, and most Chlorella had higher TAG content compared with the type strain, with only two exceptions (THX130 and HBX169). In particular, strain NJ-7, NJ-18, NMX35N and NMX139N together with THX129, HBX356 and NMX310 exhibited rather high TAG content. Of these 24 oil-producing strains, 23 belong to ‘‘true’’ Chlorella based on the phylogenetic 18S rDNA sequence analysis (Fig. 2). ‘‘C.’’ zofingiensis, ‘‘C.’’ emersonii (= Graesiella emersonii), ‘‘Chlorella’’ minutissima, ‘‘C.’’ protothecoides (= Auxenochlorella protothecoides), ‘‘C.’’ kessleri (= Parachlorella kessleri), C. sorokiniana, C. vulgaris, and Chlorella pyrenoidosa are common Chlorella species sensu lato that have been investigated for biodiesel production (Ahn et al., 2012;

NJ-18

3.1. Isolation and identification of oil-producing Chlorella

NJ-7

3. Results and discussion

Fatty acid

the dye Nile Red (Elsey et al., 2007). Algae cells were stained using the microwave-assisted procedure according to the method of Chen et al., (2011b). The final cell concentrations were OD750 = 0.20 in the staining solutions, and final concentrations of DMSO and Nile Red were 5% (v/v) and 1 lg/ml respectively.

Table 2 Contents (% of dry cell weight) and fatty acid compositions (% of total fatty acids) of total lipids and triacylglycerols of five Chlorella species cultured at 25 °C.

Fig. 3. Specific growth rates (l) of five Chlorella strains at different temperatures.

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at 38–42 °C (high-temperature strain). In addition, a ‘‘true’’ Chlorella strain, Chlorella sp. NMX37N, with the highest growth temperature of around 35 °C (Xu et al., 2012), assume an intermediate position. Table 1 summarized the upper and lower limits of growth temperature of some common Chlorella species sensu lato. Based on the phylogenetic data and TAG content, four ‘‘true’’ Chlorella strains from different clades and the type strain of this genus SAG 211-11b were selected in this study to investigate their range of growth temperature. As shown in Fig. 3, under laboratory conditions two Antarctic Chlorella strains (NJ-7 and NJ-18) grew at temperatures from 5 to 30 °C but not at 35 °C, while the two warm temperate Chlorella strains (NMX35N and NMX139N) grew at temperatures from 15 to 40 °C but not below 10 °C. As reported previously, the highest growth temperature of SAG 211-11b was 28 °C (Huss et al., 1999; Kessler, 1985). When temperatures were set below 25 °C, the specific growth rates of all the five Chlorella species tended to increase with increased temperature. It seemed that only NMX35N behaved more like a high-temperature strain. Compared

Most of the ‘‘true’’ Chlorella species are able to grow at temperatures up to 26–32 °C (normal strains), while C. sorokiniana is a heat-resistant species in this genus with an upper limit of growth

200 4

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NJ-7 NJ-18 211-11b NMX35N

1.2

OD 750

400

800

-

0.2

0.6

NO 3 (mM)

600

-

0.4

NO 3 (mM)

-

NO 3 (mM)

800

Intensity

O

35 C NMX35N

0.8

Fluorescence Intensity (a.u.)

0.6

1000

Fluorescence Intensity (a.u.)

NO3

O

5 C NJ-18

0.8

Fluorescence Intensity (a.u.)

1000

-

Fluorescence Intensity (a.u.)

O

5 C NJ-7

0.8

-

3.2. Growth temperature of high oil-producing Chlorella

NO 3 (mM)

Chen et al., 2011a; Chu et al., 2013; Li et al., 2012; Li et al., 2013; Liu et al., 2011; Mata et al., 2010; Miao and Wu, 2006). However, only C. sorokiniana, C. vulgaris and C. pyrenoidosa are ‘‘true’’ Chlorella species according to their biochemical and phylogenetic characteristics (Huss et al., 1999; Krienitzi et al., 2004). The 18S rDNA sequence comparison demonstrated the affiliation of strain NMX35N, HBX169, THX129, NMX2, and FACHB270 to C. sorokiniana CCALA 260. It was shown that strain NJ-18, NMX37N, NMX1, and HBX326 were closely related to Chlorella lobophora Andreyeva 750-I (Fig. 2). Strain NJ-7 was found to be identical to strain SAG 211-11b in 18S rDNA sequence, cell size, structure, and reproductive characteristics (Hu et al., 2008a).

0.8

0.4

0.0 5

15

20

25

30

35

40

Temperature (°C)

Fig. 4. Nitrate concentration in BG11 medium, accumulation of TAGs detected by Nile Red assay, and biomass (measured at day 18, and at day 32 for NJ-7 and NJ-18 at 5 °C) of four Chlorella strains grown at different temperatures.

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with the other four strains, NJ-18 showed a relatively better growth at temperatures ranging from 5 to 30 °C, suggesting a better temperature adaptation in this range. 3.3. Lipid content and fatty acid composition Lipid and TAG contents in strain NJ-7, NJ-18, NMX35N and NMX139N were higher than the type strain, and the highest values, 32% (lipid) and 21% (TAG) of dry cell weight (dcw), were observed in strain NJ-7 (Table 2). Most of the ‘‘true’’ Chlorella possess oil levels between 20% and 50% dependent on the culture condition (Chen et al., 2011a; Mata et al., 2010). Although lipid contents of the five strains in our study were not very high, it was convinced that higher lipid content could be obtained through the optimization of cultivation parameters. Most microalgae lipids are similar to plant oil, which is mainly composed of saturated and monounsaturated C16 and C18 fatty acid, including that of Chlorella (Ahn et al., 2012; Li et al., 2013; Liu et al., 2011; Zhou et al., 2013). The fatty acid compositions of total lipids and TAGs were similar in the five strains with C16:0, C18:1, C18:2 and aC18:3 as the main fatty acids. C18:3 content of strain NMX35N, NMX139N and NJ-18 were lower than 10% (Table 2), which meets the requirements of the European Standard EN 14214 (2004) for biodiesel production. 3.4. Lipid accumulation property during the batch culture Based on the growth temperature range and fatty acid composition, strain NMX35N, NJ-18 and NJ-7 were selected for the investigation of biomass and TAG accumulation at various temperatures with strain SAG 211-11b used as the control. As shown in Fig. 4, TAG content detected by Nile Red assay dramatically increased after nitrate concentration in the medium dropped to zero. The increasing temperature tended to enhance TAG content detected by Nile Red assay in strain NMX35N, NJ-18 and NJ-7, however, a further temperature increase to above 25 °C resulted in the decrease of the content. It was found that the optimal growth temperature for these four Chlorella strains to accumulate TAGs was 25 °C, except the type strain SAG 211-11b, whose highest TAG content detected by Nile Red assay was obtained at 15 °C. NJ-18 boasted a much higher TAG content, according to Nile Red assay, than NJ-7 at 5 °C; in particular, TAG content detected by Nile Red assay in NJ-18 almost doubled that in NJ-7 at day 32 and the biomass of NJ-18 was far higher compared with that of NJ-7. On the other hand, within the growth temperature range of 15–20 °C, the highest TAG content detected by Nile Red assay was observed in NJ-7, which was slightly higher than that in NJ-18. Meanwhile, NJ-18 possessed the highest biomass among the four strains. At 25 °C, NJ-18 and NJ-7 had the comparable TAG content detected by Nile Red assay, which was higher than other two strains, and NJ-18 exhibited the highest biomass. At 30 °C, TAG content detected by Nile Red assay in NMX35N and NJ-7 were approximately 70% of that in NJ-18. Raising temperatures from 35 to 40 °C, TAG content detected by Nile Red assay in NMX35N was almost the same, but its biomass lowered markedly. Therefore, according to the analysis of biomass, TAG content and fatty acid composition, NJ-18 could be a good candidate for biodiesel production within the growth temperature range of 5–30 °C, and MNX35N could be used as feedstock for biodiesel production at higher temperatures. Consistent with our study, it was found that the highest biomass and lipid content in ‘‘C.’’ minutissim (from 20 to 25 °C) and C. vulgaris (from 25 to 38 °C) were achieved at 25 °C (Converti et al., 2009; Seto et al., 1984). However, Li et al., (2013) showed that the lipid content of the heat-resistant C. sorokiniana increased from 21 to 30 °C and then decreased in the range of 30 to 42 °C, and the highest biomass was obtained at 37 °C. As for other microalgae, the biomass and TAG productivity in Scenedesmus sp. LX1 were al-

most the same at the temperature of 20, 25 and 30 °C (Li et al., 2011), while Converti et al., (2009) found that higher temperature (from 20 to 25 °C) was benefit for the lipid accumulation in Nannochloropsis oculata. Therefore, environmental temperature’s effect on the biomass and lipid accumulation in microorganisms including Chlorella is species/strain-specific. In addition, strain NJ-18 was reported as the best among the six oil-producing Chlorella species. This strain could be cultivated outdoors in a large scale and higher lipid productivity could be obtained through the optimization of key nutrients and cultivation methods, thus it is a potential feedstock for biodiesel (Zhou et al., 2013). It was found in our study that strain NJ-18 adapted well to a wide range of growth temperatures, which reinforced the assumption of using it for outdoor mass cultivation without temperature control. 4. Conclusion As an important environmental factor affecting algal physiology, temperature varies from below zero to above 40 °C for outdoor cultivations. Although Chlorella species can adapt to 2– 42 °C, no single strain can grow over this broad range. In this study, two high oil-producing strains, NJ-18 and NMX35N, were selected among 23 oleaginous Chlorella species from different climate zones. NJ-18 can produce high TAG content at 5–30 °C, while NMX35N can be used as the oil source at higher temperatures. The cultivation of them together is a good option for economic biodiesel production during the whole seasons of the year. Acknowledgements This work was supported by National Key Basic Research Project of China (2011CB200901) and the Open Fund Project of State Key Laboratory of Freshwater Ecology and Biotechnology (No. 2013FB15). References Ahn, J.-W., Hwangbo, K., Lee, S.Y., Choi, H.-G., Park, Y.-I., Liu, J.R., Jeong, W.-J., 2012. A new Arctic Chlorella species for biodiesel production. Bioresour. Technol. 125, 340–343. Bligh, E.G., Dyer, W.J., 1959. A rapid method of lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Borowitzka, M.A., Moheimani, N.R., 2013. Sustainable biofuels from algae. Mitig. Adapt. Strategies Glob. Change 18, 13–25. Chen, C.-Y., Yeh, K.-L., Aisyah, R., Lee, D.-J., Chang, J.-S., 2011a. Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: A critical review. Bioresour. Technol. 102, 71–81. Chen, W., Sommerfeld, M., Hu, Q., 2011b. Microwave-assisted Nile red method for in vivo quantification of neutral lipids in microalgae. Bioresour. Technol. 102, 135–141. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306. Chisti, Y., 2008. Biodiesel from microalgae beats bioethanol. Trends Biotechnol. 26, 126–131. Chu, F.-F., Chu, P.-N., Cai, P.-J., Li, W.-W., Lam, P.K.S., Zeng, R.J., 2013. Phosphorus plays an important role in enhancing biodiesel productivity of Chlorella vulgaris under nitrogen deficiency. Bioresour. Technol. 134, 341–346. Collos, Y., Mornet, F., Sciandra, A., Waser, N., Larson, A., Harrison, P.J., 1999. An optical method for the rapid measurement of micromolar concentrations of nitrate in marine phytoplankton cultures. J. Appl. Phycol. 11, 179–184. Converti, A., Casazza, A.A., Ortiz, E.Y., Perego, P., Del Borghi, M., 2009. Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production. Chem. Eng. Process: Process Intensific. 48, 1146–1151. de-Bashan, L.E., Trejo, A., Huss, V.A.R., Hernandez, J.-P., Bashan, Y., 2008. Chlorella sorokiniana UTEX 2805, a heat and intense, sunlight-tolerant microalga with potential for removing ammonium from wastewater. Bioresour. Technol. 99, 4980–4989. Elsey, D., Jameson, D., Raleigh, B., Cooney, M.J., 2007. Fluorescent measurement of microalgal neutral lipids. J. Microbiol. Methods 68, 639–642. European Standard EN, (2004). Automotive fuels-fatty acid methyl esters (FAME) for diesel engines-requirements and test methods. AFNOR, Saint-Denis. Hoek, C., Mann, D.G., Jahns, H.M., 1995. Algae: An Introduction to Phycology. Cambridge University Press, Cambridge, UK, p. 623.

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