Accepted Manuscript

Feasibility of biodiesel production by microalgae Chlorella sp. (FACHB-1748)

under outdoor conditions

XuPing Zhou, Ling Xia, HongMei Ge, DeLu Zhang, ChunXiang Hu

PII: S0960-8524(13)00573-7

DOI: http://dx.doi.org/10.1016/j.biortech.2013.03.169

Reference: BITE 11629

To appear in: Bioresource Technology

Received Date: 5 February 2013

Revised Date: 23 March 2013

Accepted Date: 26 March 2013

Please cite this article as: Zhou, X., Xia, L., Ge, H., Zhang, D., Hu, C., Feasibility of biodiesel production by

microalgae Chlorella sp. (FACHB-1748) under outdoor conditions, Bioresource Technology (2013), doi: http://

dx.doi.org/10.1016/j.biortech.2013.03.169

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1

Feasibility of biodiesel production by microalgae Chlorella sp. (FACHB-1748)

under outdoor conditions

XuPing Zhoua,b, Ling Xiaa,b, HongMei Gea,b, DeLu Zhangc, ChunXiang Hua,b

aKey Laboratory of Algal Biology, Institute of Hydrobiology, University of Chinese

Academy of Sciences, Wuhan 430072, China

bUniversity of Chinese Academy of Sciences, Beijing 100049, China

cDepartment of Biological Science and Biotechnology, Wuhan University of

Technology, Wuhan 430070, China

*Corresponding author: Tel./fax: +86 27 68780866

E-mail address: cxhu@ihb.ac.cn. (C.X. Hu)

2

Abstract

Chlorella sp. (FACHB-1748) was cultivated outdoors under natural sunlight to evaluate

its potential for biofuel production. Urea was selected as nitrogen source, and the

concentration was optimized. When the culture reached the late exponential stage, a

triggering lipid accumulation test was conducted using different concentrations of

sodium chloride and acetate. A scaling-up experiment was also conducted in a 70 L

photobioreactor. The highest biomass productivity (222.42, 154.48 mg/L/d) and lipid

productivity (64.30, 33.69 mg/L/d) were obtained with 0.1 g/L urea in 5 and 70 L

bioreactors, respectively. The highest lipid content (43.25%) and lipid yield (1243.98

mg/L) were acquired with the combination of 10 g/L sodium chloride and acetate.

Moreover, the qualities of biodiesel, cetane number, saponification value, iodine value,

and cold filter plugging point complied with the standards set by the National Petroleum

Agency (ANP255), Standard ASTMD6751, and European Standard (EN 14214).

Keywords: Acetate; Biodiesel; Sodium chloride; Urea; Outdoor condition

1. Introduction

Microalgae have been considered as a promising alternative source for biodiesel

3

production because of their photosynthetic efficiency, high lipid content, and ability to

grow in extreme environments (Hu et al., 2008; Courchesne et al., 2009). However,

reducing the cost of biodiesel production, improving lipid content, and maintaining

selected species under natural sunlight in outdoor culture are some of the major

obstacles for commercial production (Rodolfi et al., 2008).

Nitrogen is one of the major nutrients required for algal growth. Various

microalgae, including Spirulina platensis (Soletto et al., 2005; Matsudo et al., 2009),

Neochloris oleoabundans (Li et al., 2008), Scenedesmus dimorphus (Shen et al., 2009a),

and Chlorella sp. (Hsieh and Wu, 2009; Shen et al., 2009b; Perez-Garcia et al., 2011),

can grow well with urea as nitrogen source. In this study, we cultivated the alga using

an alternative low cost nitrogen source, which can be advantageous for commercial

production.

Many studies focused on improving the lipid content of microalgae under nutrient

conditions, such as nitrogen, phosphorous, silicon starvation, and salt stress (Lynn et al.,

2000; Khozin-Goldberg and Cohen, 2006; Li et al., 2010; Herrera-Valencia et al., 2011;

Kaewkannetra et al., 2012). However, growth is inhibited heavily under such conditions

because of low lipid productivity.

Most studies on biodiesel production by microalgae have been conducted in the

laboratory. Only a few studies focused on outdoor cultivation. Microalgae cultured

outdoors can utilize natural sunlight to grow, thereby reducing the overall cost of

biodiesel production from algae (Oh et al., 2010; Feng et al., 2012). Hence, the

4

feasibility of cultivating microalgae under outdoor conditions should be tested.

In this study, all experiments were conducted under outdoor environment. Urea was

selected as nitrogen source because of its low cost, and the concentration was optimized

for the potential oleaginous microalga Chlorella sp. When the culture reached the late

exponential stage, different concentrations of sodium chloride and acetate were added to

stimulate lipid accumulation. Important fuel properties of biodiesel were also analyzed

for the said microalga, and large-scale cultivation was conducted in a 70 L

photobioreactor under outdoor conditions.

2. Materials and methods

2.1. Strain and cultivation conditions

Chlorella sp. (FACHB-1748) isolated from a pond in Huang Gang, China was cultured

in BG11 medium containing the following components: 1.5 g/L NaNO3, 40 mg/L

KH2PO4·3H2O, 75 mg/L MgSO4·7H2O, 36 mg/L, CaCl2·2H2O, 6.0 mg/L citric acid, 6.0

mg/L ferric ammonium citrate, 1.0 mg/L EDTA, 20 mg/L Na2CO3, and 1.0 mL/L A5

solution. In this study, however, we substituted urea for NaNO3. All reagents (purity

>99.5%) were provided by Sinopharm Chemical Reagent Co., Ltd.

This study was conducted in Beijing, China (40°22′N, 116°20′E). Triangular flasks

(5 L) were used to optimize urea concentrations and induce lipid accumulation. A 70 L

photobioreactor (22 cm diameter, 185 cm height) provided by Dalian Huixin Titanium

5

Equipment Development, Co., Ltd. was used to test whether the microalgal sample can

grow well in large-scale culture conditions. The material is heat resistant with

light-pervious nylon membrane.

The concentrations for the urea optimization experiment were set as 0, 0.1, 0.25,

and 0.5 g/L.

For induction of lipid accumulation, 0, 5, and 10 g/L sodium chloride and 10 g/L

acetate were added when the culture reached the late exponential stage.

2.2. Determination of biomass, total lipid fatty acid content, and quality of biodiesel

The biomass (dry weight, DW) was determined spectrophotometrically by

TU-1900 UV spectroscopy at 680 nm (OD680). Biomass was then calculated by

multiplying OD680 values with 0.38, a predetermined conversion factor to convert the

OD680 value to dry weight.

Approximately 50 mg to 100 mg of dried algal sample was lyophilized using a

vacuum freeze dryer (Alpha 1-2 LD plus; Christ). Total lipid was extracted from the

dried alga sample using a Soxhlet apparatus, with chloroform-methanol (2:1, v/v) as

solvent (Cheung et al., 1998; Hsieh and Wu, 2009; Zhou et al., 2013). Fatty acid

components were analyzed by gas chromatography–mass spectrometry (Ultra Trace,

Thermo-Scientific, USA) equipped with a DB-23 capillary column (0.25 mm × 60 m;

0.25 mm, film thickness; Agilent Technologies, USA) and an FID detector. The initial

6

temperature was maintained at 50 °C for 1 min and then increased to 170 °C at a rate of

40 °C/min, with each temperature increment kept for 1 min. The temperature was raised

from 170 °C to 210 °C at 18 °C/min, which was maintained for 28 min. The injector

temperature was 270 °C, and the split ratio was 50:1. Nitrogen was used as a carrier gas

with a flow rate of 2.0 mL/min. The detector temperature was 280 °C, and air and

hydrogen flows were set at 350 and 35 mL/min, respectively. A 1 μL sample was

injected for analysis. The internal standard used was Margaric acid triglycerides (C17:0),

and the calculation method was area normalization.

The key qualities of biodiesel, iodine value (IV), saponification value (SV), cetane

number (CN), long-chain saturated factor (LCSF), and cold filter plugging point (CFPP)

were calculated using the following formulas (Ramos et al., 2009; Francis et al., 2010):

IV = M

)ND254(∑ ××

SV = M

)N560(∑ ×3

CN = )IV225.0(SV

54583.46 ×−+

LCSF = (0.1×C16) + (0.5×C18) + (1× C20) + (1.5 ×C22) + (2×C24)

CFPP = 3.1417 ×LCSF－16.477

where D, N, M, are the number of double bonds, the percentage of each fatty acid

component, and the molecular mass, respectively. C16, C18, C20, C22, and C24 are the

weight percentages of each of the fatty acids (wt%) (Ramos et al., 2009; Francis et al.,

2010).

7

3. Results and discussion

3.1. Biomass and lipid accumulation of Chlorella sp. at different urea concentrations

In this study, the effects of different urea concentrations on the growth and lipid content

of Chlorella sp. under outdoor conditions were investigated. As shown in Table 1, the

highest biomass productivity (222.42 mg/L/d) was obtained at the concentration of 0.1

g/L. Cell growth was found to be inversely proportional to urea and was inhibited

significantly under nitrogen-deficient conditions, with urea concentrations ranging from

0.25 to 0.5 g/L (Fig. 1). Biomass productivity was only 42.71 mg/L/d under urea

starvation, and it decreased to 138.46 mg/L/d at 0.5 g/L urea (Table 1). This finding

may be attributed to the spontaneous hydrolysis of urea to ammonia and to the toxicity

of high ammonia concentration to algal growth (Danesi et al., 2002).

Lipid content was also affected by urea concentration. As shown in Table 1, the highest

lipid content (34.13%) was observed under urea-limited conditions, and lipid content

gradually decreased with increasing urea concentration. It was only 23.11% at 0.5 g/L

urea. Lipid productivity was the lowest (14.79 mg/L/d) under urea starvation and the

highest (64.30 mg/L/d) at 0.1 g/L urea. Griffiths and Harrison (2009) suggested that

lipid productivity is the key parameter in selecting algal species for biodiesel

production. Hsieh and Wu (2009) reported the highest lipid productivity of 124 mg/L/d

8

for Chlorella sp. at 0.1 g/L urea. This value was higher than the result obtained in our

study. However, the photobioreactor used in the present experiment was five times

larger than what they employed. More importantly, the strain used in the present study

was cultivated outdoors under natural sunlight. Most experiments on biodiesel

production from algae were conducted in the laboratory, with fluorescent lamp as light

source. Hence, cultivation of microalgae under direct sunlight can dramatically reduce

the cost of industrial biodiesel production (Oh et al., 2010; Feng et al., 2012). Feng et al.

(2012) cultured Chlorella zofingiensis in a 10 L photobioreactor under outdoor

conditions and obtained only a maximum biomass productivity of 36 mg/L/d. This

value is just 0.16 times that of Chlorella sp. (222.42 mg/L/d) in the present study (Table

1). These results suggest that Chlorella sp. has great potential for biodiesel production

and that 0.1 g/L urea is the optimal concentration for its growth and lipid accumulation.

3.2. Biomass, lipid accumulation, and biodiesel qualities of Chlorella sp. under

different concentrations of sodium chloride and acetate

Many studies investigated the effects of different NaCl concentrations on the

oil-producing alga. Lipid content was observed to increase dramatically under high

NaCl concentrations. Kaewkannetra et al. (2012) reported that the lipid content of

Scenedesmus obliquus improved from 9.5% to 36% with 0.3 M NaCl. Herrera-Valencia

et al. (2011) also found that the lipid productivity of Chlorella saccharophila can be

9

improved from 63.3 mg/L/d to 79 mg/L/d with 0.15 M NaCl. However, the biomass was

significantly lower than that of the control without salt. In our previous studies, NaCl

was added at the initial inoculation stages. Hence, microalgal growth was dramatically

inhibited at high salt concentrations, resulting in relatively low biomass (data not

shown). In the present study, the sample strain was cultured in an optimized medium to

initially accumulate high biomass. When the culture reached the exponential stage, high

concentrations of NaCl and acetate were added to induce lipid accumulation.

Chlorella sp. was cultured using the optimal urea concentration (0.1 g/L)

determined from a previous study, and high concentrations of salt and acetate were

added into the culture to induce lipid accumulation. The results are summarized in Table

2. The dry weight (3.19 g/L) was the highest in 5 g/L sodium chloride on day 13, and

the biomass of the treatments with both acetate and sodium chloride or acetate alone

was higher than that of the control on day 10. However, the dry weight of the samples

treated with only 10 g/L sodium chloride was lower than that of the control (Fig. 2 and

Table 2). The results indicate that 5 g/L sodium chloride and 10 g/L acetate can promote

the growth of the algal sample. However, >5 g/L sodium chloride can inhibit it. Takagi

et al. (2006) reported that the marine microalga Dunaliella can grow well in NaCl

concentrations less than 1.0 M (58.5 g/L), which might be due to its salt-tolerant

characteristics. Lipid content increased with increasing salt concentration, and all the

treatments had higher lipid contents than the control. The highest lipid content (40.34%)

10

was obtained under 10 g/L sodium chloride and acetate on day 10 (Table 2). On day 13,

the highest lipid content (43.25%) and lipid yield (1243.98 mg/L) were obtained under

the b10 (AC) +10 (SC) treatment (Table 2).

The fatty acid profiles are shown in Table 3. More than 90% of the profiles were

C16－C18, which are known to be the predominant components of biodiesel (Li et al.,

2010). Higher proportion of C18:1 was obtained with increasing NaCl (Table 3), which

was 43.93% and 44.17% under a10 (SC) +10 (AC) and b10 (AC) +10 (SC), respectively.

C18:3 increased from 16.23% to 21.63% with increasing NaCl but decreased to 11.95%

in treatments with the combination of sodium chloride and acetate (Table 3).

Several important parameters (CN, SV, IV, and CFPP) of biodiesel production were

investigated in this study. The results are summarized in Table 4. CN is the indicator of

ignition and combustion quality, with the minimum standard values of 45, 47, and 51

according to the National Petroleum Agency (ANP255), Standard ASTMD6751, and

European Standard (EN 14214), respectively (Francisco et al. 2010). The values (49.11

to 52.70) obtained for all the treatments in this study were in accordance with the

aforementioned standards (Table 4). IV is another parameter of biodiesel production,

which is limited to 120 g I2 100 g -1 by the European standard (EN 14214). Therefore,

the IV (95.33 to 112.03) of Chlorella sp. in this work is suitable for the said purpose.

CFPP, which indicates the flow performance of biodiesel at low temperature, was

approximately -15 °C for the algal sample (Table 4). The temperature limits are

different for each country and climate, with 0 and -10 °C being the maximum limits for

11

summer and winter in Spain, respectively (Knothe, 2006; Ramos et al., 2009). The

biodiesel quality of the algal sample complied with the standards set by the National

Petroleum Agency (ANP255), Standard ASTMD6751, and European Standard (EN

14214).

3.3. Biomass and lipid accumulation of Chlorella sp. in a 70 L photobioreactor

To test the feasibility of large-scale cultivation under outdoor conditions, outdoor

cultures of Chlorella sp. were initiated and maintained in a 70 L photobioreactor. The

culture medium was BG 11, using 0.1 g/L urea (obtained in previous experiment in

section 3.1) as substitute for NaNO3. The cells grew well and achieved high biomass

productivity in the large photobioreactor. As shown in Fig. 3, 154.48 mg/L/d of biomass

productivity and 21.27% of lipid content were obtained on day 7. The biomass

productivity (154.48 mg/L/d) was 2.65 times higher than that of Chlorella zofingiensis

(58.4 mg/L/d) cultured outdoor in a 60 L flat photobioreactor (Feng et al., 2011). Zheng

et al. (2012) and Oh et al. (2010) found that the lipid contents of Chlorella sorokiniana

and Chlorella minutissima were 13.1% and 12.8%, respectively, under autotrophic

conditions in large photobioreactors. These values are much lower than that obtained

from Chlorella sp. (21.27%) in this study. Therefore, Chlorella sp. can be a potential

feedstock for biodiesel production.

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4. Conclusions

The oleaginous alga Chlorella sp. (FACHB-1748) can be cultured with urea as

nitrogen source in large photobioreactors under outdoor conditions. Furthermore, lipid

content was significantly improved under the induction of sodium chloride and acetate.

The quality and properties of the biodiesel produced met the criteria of the National

Petroleum Agency (ANP255), Standard ASTMD6751, and European Standard (EN

14214). The results demonstrated the utility of Chlorella sp. (FACHB-1748) as a

potential feedstock for biodiesel production.

Acknowledgments

The authors acknowledge the financial support provided by the Program of Sinopec

(Y149121601), National 863 Program (2013AA065804), International Partner Program

of Innovation Team (University of Chinese Academy of Sciences), and the Platform

Construction of Oleaginous Microalgae (Institute of Hydrobiology, UCAS of China).

13

References

(1) Cheung, P.C.K., Leung, A.Y.H., Ang, P.O.A., 1998. Comparison of Supercritical

Carbon Dioxide and Soxhlet Extraction of Lipids from a Brown Seaweed, Sargassum

hemiphyllum (Turn.) C. Ag. J. agric. Food Chemi. 46, 4228–4232.

(2) Courchesne, N.M.D., Parisien, A., Wang, B., Lan, C.Q., 2009. Enhancement of

lipid production using biochemical, genetic and transcription factor engineering

approaches. J. Biotechnol. 141, 31–41.

(3) Danesi, E.D.G., Rangel-Yagui, C.O., Carvalho, J.C.M., Sato, S., 2002. An

investigation of effect of replacing nitrate by urea in the growth and production of

chlorophyll by Spirulina platensis. Biomass. Bioen. 23, 261–269.

(4) Feng, P.Z., Deng, Z.Y., Hu, Z.Y., Fan, L., 2011. Lipid accumulation and growth of

Chlorella zofingiensis in flat plate photobioreactors outdoors. Bioresour. Technol.

102, 10577–10584.

(5) Feng, P.Z., Deng, Z.Y., Fan, L., Hu, Z.Y., 2012. Lipid accumulation and growth

characteristics of Chlorella zofingiensis under different nitrate and phosphate

concentrations. J. Biosci. Bioeng. 102, 106–110.

(6) Francisco, E.C., Neves, D.B., Jacob-Lopes, E., Franco, T.F., 2010. Microalgae as

feedstock for biodiesel production: Carbon dioxide sequestration, lipid production

and biofuel quality. J. Chem. Technol. Biotechnol. 85, 395–403.

(7) Griffiths, M.J., Harrison, S.T.L., 2009. Lipid productivity as a key characteristic for

14

choosing algal species for biodiesel production. J. Appl. Phycol. 21, 493–507.

(8) Herrera-Valencia, V,A., Contreras-Pool, P.Y., Lopez-Adrian, S.J.,

Peraza-Echeverrıa, S, Barahona-Perez, L.F., 2011. The green microalga Chlorella

saccharophila as a suitable source of oil for biodiesel production. Curr. Microbiol.

63, 151–157.

(9) Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M.,

Darzins, A., 2008. Microalgal triacylglycerols as feedstocks for biofuel

production: perspectives and advances. Plant. J. 54, 621-639.

(10) Hsieh, C.H., Wu, W.T., 2009. Cultivation of microalgae for oil production with a

cultivation strategy of urea limitation. Bioresour. Technol. 100, 3921–3926.

(11) Kaewkannetra, P., Enmak, P., Chiu, T.Z., 2012. The effect of CO2 and salinity on

the cultivation of Scenedesmus obliquus for biodiesel production. Biotechnol.

Bioprocess Eng. 17, 591–597.

(12) Khozin-Goldberg, I., Cohen, Z., 2006. The effect of phosphate starvation on the

lipid and fatty acid composition of the fresh water eustigmatophyte Monodus

Subterraneus. Phytochem. 67, 696–701.

(13) Knothe, G., 2006. Analyzing biodiesel: standards and other methods. J. Am. Oil

Chem. Soc. 83, 823–833.

(14) Li, Y., Horsman, M., Wang, B., Wu, N., Lan, C.Q., 2008. Effects of nitrogen

sources on cell growth and lipid accumulation of green alga Neochloris

oleoabundans. Appl. Microbio. Biotechnol. 81, 629-636.

15

(15) Li, X., Hu, H.Y., Gan, K., Sun, Y.X., 2010. Effects of different nitrogen and

phosphorus concentrations on the growth, nutrient uptake, and lipid accumulation of

a freshwater microalga Scenedesmus sp. Bioresour. Technol. 101, 5494–5500.

(16) Lynn, S.G., Kilham, S.S., Kreeger, D.A., Interlandi, S.J., 2000. Effect of nutrient

availability on the biochemical and elemental stoichiometry in fresh water diatom

Stephanodiscus minutulus (bacillariophyceae). J. Phycol. 36, 510–522.

(17) Matsudo, M.C., Bezerra, R.P., Sato, S., Perego, P., Converti, A., Carvalho, J.C.M.,

2009. Repeated fed-batch cultivation of Arthrospira (Spirulina) platensis using urea

as nitrogen source. Biochem. Eng. J. 43, 52–57.

(18) Oh, S.H., Kwon, M.C., Choi, W.Y., Seo, Y.C., Kim, G.B., 2010. Long-term outdoor

cultivation by perfusing spent medium for biodiesel production from Chlorella

minutissima. J. Biosci. Bioeng. 110, 194–200.

(19) Perez-Garcia, O., Escalante, F.M.E., de-Bashan, L.E., Bashan, Y., 2011.

Heterotrophic cultures of microalgae: metabolism and potential products. Water res.

45, 11–36.

(20) Ramos, M.J., Feranandez, G.M., Casas, A., Rodriguez, L., Perez, A., 2009. Influence

of fatty acid composition of raw materials on biodiesel properties. Bioresour.

Technol. 100, 261–268.

(21) Rodolfi, L., Zittelli, G.C., Bassi, N., 2008. Microalgae for oil: Strain selection,

induction of lipid synthesis and outdoor mass cultivation in a low-cost

photobioreactor. Biotechnol. Bioeng. 102, 100–112.

16

(22) Shen, Y., Pei, Z.J., Yuan, W.Q., Mao, E.R., 2009a. Effect of nitrogen and extraction

method on algae lipid yield. Int. Agric. Biol. Eng. 2, 51–57.

(23) Shen, Y., Yuan, Z., Pei, Z., Mao, E., 2009b. Heterotrophic Culture of Chlorella

protothecoides in Various Nitrogen Sources for Lipid Production. Appl. Biochem.

Biotechnol. 160, 1674–1684.

(24) Soletto, D., Binaghi, L., Lodi, A., Carvalho, J.C.M., Converti, A., 2005. Batch and

fed-batch cultivation of Spirulina platensis using ammonium sulphate and urea as

nitrogen sources. Aquac. 243, 217–224.

(25) Takagi, M., Karseno, Yoshida, T., 2006. Effect of salt concentration on

Intracellular accumulation of lipids and triacylglyceride in marine microalgae

Dunaliella cells. J. Biosci. Bioeng. 101, 223–226.

(26) Zheng, Y.B., Chi, Z.Y., Lucker, B., Chen, S.L., 2012. Two-stage heterotrophic

and phototrophic culture strategy for algal biomass and lipid production.

Bioresour. Technol. 102, 484–488.

(27) Zhou, X.P, Ge, H.M, Xia, L., Zhang, D., Hu, C.X, 2013. Evaluation of oil-producing

algae as potential biodiesel feedstock. Bioresour. Technol. 134, 24–29.

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Table Captions

Table 1 Biomass productivity, lipid content and lipid productivity of Chlorella sp.

under different concentrations of urea

Table 2 Lipid content of Chlorella sp. under different concentrations of sodium

chloride and acetate

Table 3 Fatty acids of Chlorella sp. on 13th day under different concentrations (g/L)

of sodium chloride and acetate

Table 4 Quality properties of the biodiesel under different concentrations (g/L) of

sodium chloride and acetate

Figure captions

Figure 1

Dry weight of Chlorella sp. under different urea concentrations

Figure 2

Dry weight of Chlorella sp. under different sodium chloride and acetate concentrations

(Arrow indicates the time sodium chloride and acetate added. a10 g/L sodium chloride

and 10 g/L acetate added on day 8; b10 g/L acetate added on day 8 and 10 g/L sodium

chloride added on day 10)

Figure 3

Dry weight, lipid content and lipid productivity of Chlorella sp. in 70 L

photobioreactors under outdoor condition (DW, Dry weight (g/L); LC, Lipid content

(%); LP, Lipid productivity (mg/L/d))

18

Table 1 Biomass productivity, lipid content and lipid productivity of Chlorella sp.

under different concentrations of urea

Concentration

(g/L)

Biomass Productivity

(mg/L/d)

Lipid Content

(%)

Lipid Productivity

(mg/L/d)

0 42.71±1.21 34.13±0.15 14.79±0.27

0.1 222.42±6.64 28.48±0.49 64.30±2.04

0.25 204.50±7.29 27.03±1.40 56.31±3.90

0.5 138.46±2.93 23.11±0.32 46.06±0.15

19

Table 2 Lipid content of Chlorella sp. under different concentrations of sodium chloride and acetate

SC, sodium chloride; AC, acetate a10 g/L sodium chloride (SC) and 10 g/L acetate (AC) added on day 8; b10 g/L acetate added on day 8 and 10 g/L sodium chloride added

on day 10, the same below.

Concentration

(g/L)

8 day 10 day 13 day

Dry weight

(g/L)

Lipid content

(%)

Dry weight

(g/L)

Lipid content

(%)

Dry weight

(g/L)

Lipid content

(%)

Lipid yield

(mg/L)

0 2.13±0 28.91±0 2.65±0.00 29.25±0.30 2.95±0.13 33.49±1.60 988.28±5.14

5 (SC) 2.13±0 28.91±0 2.96±0.05 32.18±1.53 3.19±0.01 36.74±2.28 1171.42±5.03

10 (SC) 2.13±0 28.91±0 2.59±0.04 35.10±1.27 2.68±0.16 37.01±0.70 993.33±3.21 a10(SC)+10 (AC) 2.13±0 28.91±0 2.86±0.09 40.34±1.82 2.74±0.04 41.12±3.52 1127.84±7.11 b10(AC)+10 (SC) 2.13±0 28.91±0 2.75±0.15 32.32±1.31 2.88±0.14 43.25±1.73 1243.98±2.09

20

Table 3 Fatty acids of Chlorella sp. on 13th day under different concentrations

(g/L) of sodium chloride and acetate

ND: not detected

0 5 (SC) 10 (SC) a10(SC)+10(AC) b10(AC)+10(SC)

C14:0 3.59±0.28 1.02±0.00 0.82±0.03 0.44±0.02 0.64±0.01

C14:1 0.17±0.00 0.05±0.00 0.06±0.00 0.03±0.00 0.06±0.00

C16:0 29.53±2.13 28.29±2.34 26.76±114 25.10±3.19 23.98±1.09

C16:1 0.25±0.00 0.19±0.00 0.02±0.00 0.02±0.00 0.05±0.00

C18:0 4.00±0.02 3.97±0.11 3.48±0.02 4.27±0.11 4.61±0.09

C18:1 29.18±2.21 30.78±2.93 30.57±2.36 43.93±2.54 44.17±4.32

C18:2 15.18±0.49 15.81±2.23 16.40±1.34 15.32±0.20 14.67±0.93

C18:3 16.23±2.08 19.24±1.32 21.63±4.23 11.34±3.01 11.95±1.00

C18:4 0.50±0.00 0.42±0.00 0.31±0.00 0.41±0.00 0.42±0.00

C20:0 0.06±0.00 0.03±0.00 0.01±0.00 0.05±0.00 0.08±0.00

C20:3 0.30±0.00 0.01±0.00 ND ND ND

C20:4 0.01±0.00 0.06±0.00 ND ND ND

C22:0 1.00±0.00 0.13±0.00 0.03±0.00 0.07±0.00 0.01±0.00

21

Table 4 Quality properties of the biodiesel under different concentrations (g/L) of

sodium chloride and acetate

CN, cetane number (wt%); SV, saponification value; IV, iodine value (g I2100 g−1);

LCFS, long-chain saturated factor (wt%); CFPP, cold filter plugging point (°C)

0 5 (SC) 10 (SC) a10 (SC)+10 (AC) b10(AC)+10 (SC)

CN 51.94 50.16 49.11 52.70 52.68

SV 196.04 195.74 194.83 195.97 195.20

IV 98.65 106.78 112.03 95.33 95.91

LCSF 0.38 0.38 0.39 0.40 0.40

CFPP －15.30 －15.27 －15.26 －15.21 －15.21

22

Figure 1

Dry weight of Chlorella sp. under different urea concentrations

23

Figure 2

Dry weight of Chlorella sp. under different sodium chloride and acetate

concentrations (Arrow indicates the time sodium chloride and acetate added. a10 g/L

sodium chloride and 10 g/L acetate added on day 8; b10 g/L acetate added on day 8

and 10 g/L sodium chloride added on day 10)

24

Figure 3

Dry weight, lipid content and lipid productivity of Chlorella sp. in 70 L

photobioreactors under outdoor condition (DW, Dry weight (g/L); BP, biomass

productivity (mg/L/d); LC, Lipid content (%); LP, Lipid productivity (mg/L/d))

25

Highlights

Chlorella sp. (FACHB-1748) could utilize urea as nitrogen source.

Chlorella sp. could be cultured in 70 L bioreactor with natural sunlight under

outdoor conditions.

The lipid productivity could be improved under the condition of sodium chloride

or acetate.

The quality properties of the biodiesel met the standard of National Petroleum

Agency (ANP255).