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:https://www.doczj.com/doc/c512512307.html,/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:// https://www.doczj.com/doc/c512512307.html,/10.1016/j.biortech.2013.03.169
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Feasibility of biodiesel production by microalgae Chlorella sp.(FACHB-1748) under outdoor conditions
XuPing Zhou a,b, Ling Xia a,b, HongMei Ge a,b, DeLu Zhang c, ChunXiang Hu a,b a Key Laboratory of Algal Biology, Institute of Hydrobiology, University of Chinese Academy of Sciences, Wuhan 430072, China
b University of Chinese Academy of Sciences, Beijing 100049, China
c Department of Biological Science an
d Biotechnology, Wuhan University of Technology, Wuhan 430070, China
*Corresponding author: Tel./fax: +86 27 68780866
E-mail address: cxhu@https://www.doczj.com/doc/c512512307.html,. (C.X. Hu)
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
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
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
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
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
weight percentages of each of the fatty acids (wt%) (Ramos et al., 2009; Francis et al., 2010).
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
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
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%)
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
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.
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).
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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 4Quality 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))
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