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: [email protected]. (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.
12
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
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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
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).