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Effects of cultivation conditions and media composition on cell growth and lipid productivity of indigenous microalga Chlorella vulgaris ESP-31
Bioresource Technology 105 (2012) 120–127
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Effects of cultivation conditions and media composition on cell growth and lipid productivity of indigenous microalga Chlorella vulgaris ESP-31 Kuei-Ling Yeh a, Jo-Shu Chang a,b,c,⇑ a
Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan c Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan, Taiwan b
a r t i c l e
i n f o
Article history: Received 1 October 2011 Received in revised form 23 November 2011 Accepted 26 November 2011 Available online 2 December 2011 Keywords: Chlorella vulgaris Lipid productivity Microalgal lipid Medium composition Cultivation condition
a b s t r a c t The growth and lipid productivity of an isolated microalga Chlorella vulgaris ESP-31 were investigated under different media and cultivation conditions, including phototrophic growth (NaHCO3 or CO2, with light), heterotrophic growth (glucose, without light), photoheterotrophic growth (glucose, with light) and mixotrophic growth (glucose and CO2, with light). C. vulgaris ESP-31 preferred to grow under phototrophic (CO2), photoheterotrophic and mixotrophic conditions on nitrogen-rich medium (i.e., Basal medium and Modified Bristol’s medium), reaching a biomass concentration of 2–5 g/l. The growth on nitrogen-limiting MBL medium resulted in higher lipid accumulation (20–53%) but slower growth rate. Higher lipid content (40–53%) and higher lipid productivity (67–144 mg/l/d) were obtained under mixotrophic cultivation with all the culture media used. The fatty acid composition of the microalgal lipid comprises over 60–68% of saturated fatty acids (i.e., palmitic acid (C16:0), stearic acid (C18:0)) and monounsaturated acids (i.e., oleic acid (C18:1)). This lipid composition is suitable for biodiesel production. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Microalgal biomass, containing lipids, starch, cellulose, proteins, and so on, is considered a promising feedstock for producing a variety of renewable fuels, such as biodiesel, bioethanol, biohydrogen and methane (Carioca, 2010; Chisti, 2008; Posten and Schaub, 2009). Microalgal lipids have attracted much attention as future raw materials for biodiesel synthesis due to the potential of attaining much higher lipid productivity than is possible with other lipidbased energy crops (Chisti, 2007; Griffiths and Harrison, 2009). Several strategies have been applied to improve microalgae growth and lipid content. These include optimization of the medium compositions (e.g., type of carbon source, vitamins, salts and nutrients), physical parameters (e.g., pH, temperature and light intensity), and type of metabolism (e.g., phototrophic, heterotrophic, mixotrophic and photoheterotrophic growth) (Chojnacka and Marquez-Rocha, 2004; Mata et al., 2010). In particular, the type of cultivation method, using different energy sources (light or organic) and carbon sources (inorganic or organic), is always recognized as a key factor that significantly influences the growth and lipid accumulation of microalgae (Chen et al., 2011; Mata et al., 2010). ⇑ Corresponding author at: Department of Chemical Engineering, National Cheng Kung University, Tainan 710, Taiwan. Tel.: +886 6 2757575x62651; fax: +886 6 2357146. E-mail address: [email protected] (J.-S. Chang). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.11.103
Photoheterotrophic conditions are rarely mentioned in the literature, and it is usually confusing to distinguish mixotrophic from photoheterotrophic growth (Griffiths and Harrison, 2009; Mata et al., 2010). In photoheterotrophic cultivation, the microalgae require light as an energy source while using organic materials as the carbon source. In contrast, both organic carbon and CO2 are essential carbon sources in mixotrophic growth, as a light source is also supplied (Chojnacka and Marquez-Rocha, 2004). Although photoautotrophic conditions are commonly used in microalgae cultivation, some reports noted that higher biomass and lipid productivity of Chlorella sp. could be obtained under heterotrophic and mixotrophic conditions (Chen et al., 2011; Cheng et al., 2009; Li et al., 2007; Liang et al., 2009; Xiong et al., 2008). The nitrogen source and concentration in the medium used are also known two of the most crucial factors affecting the lipid content of microalgae (Hsieh and Wu, 2009; Yeh and Chang, 2011). Therefore, it is of importance to utilize appropriate cultivation conditions and medium composition (especially nitrogen source and concentration) to achieve the best lipid production performance of microalgae species. In this study, an indigenous microalgal isolate (identified as Chlorella vulgaris ESP-31) was examined with regard to its effectiveness for biomass and lipid production under different medium and cultivation conditions. To identify the lipid production performance of C. vulgaris ESP-31, the lipid content and lipid productivity were monitored with respect to time when the
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microalga was grown under different medium and cultivation conditions. The fatty acid profile of the microalgal lipid was also determined to evaluate the feasibility of using the produced microalgal lipid for biodiesel synthesis. This study aimed to identify the best conditions for the cell growth and lipid accumulation of C. vulgaris ESP-31, and to evaluate the potential of using the microalgal biomass thus produced as biodiesel feedstock. 2. Methods 2.1. Microalga strain and culture medium C. vulgaris ESP-31 was isolated from a shrimp-culturing pond located in South Taiwan. The freshwater sample collected from Southern Taiwan was inoculated into BG11 medium and grown at 25 °C in an artificial light cabinet under illumination with fluorescent lamps (60 lmol/m2 s, light/dark cycle: 16/8 h). The culture was then spread on BG11 medium agar plates and the appeared colonies were subcultured into the same medium until the pure strain was obtained (Ho et al., 2010). The identity of the microalgal isolate was determined through plastid 23S rRNA gene sequence analysis. The genomic DNA of the microalga was extracted using the Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA). The plastid 23S rRNA gene was amplified by polymerase chain reaction (PCR) using two universal algal primers reported previously (Ho et al., 2010). The sequence analysis was conducted using a DNA sequencer (ABI Prism 310; Applied Biosystems) and the DNA sequences were assembled using the Fragment Assembly System program from the Wisconsin package version 9.1. The sequences of the microalgal strains were compared against plastid 23S rRNA gene sequences available from the GenBank databases. Multiple sequence alignment including microalgal strains and their closest relatives was performed using BioEdit software and MEGA4 (Ho et al., 2010). The medium used for the preculture of C. vulgaris ESP-31 was Basal medium (Shi et al., 1997) that consisted of (g/l): KNO3, 1.25; KH2PO4, 1.25; MgSO47H2O, 1; CaCl2, 0.0835; H3BO3, 0.1142; FeSO47H2O, 0.0498; ZnSO47H2O, 0.0882; MnCl24H2O, 0.0144; MoO3, 0.0071; CuSO45H2O, 0.0157; Co(NO3)26H2O, 0.0049; EDTA2Na, 0.5. The microalga was regularly grown at 25 °C for 4–5 days with a continuous supply of 2% CO2 at an aeration rate of 0.2 vvm. The microalga culture was illuminated all day with a light intensity of ca. 60 lmol/m2 s. 2.2. Operation of photobioreactor and culture conditions The photobioreactor (PBR) used to cultivate C. vulgaris ESP-31 was a 1-liter glass vessel (15.5 cm in length and 9.5 cm in diameter) equipped with an external light source (14 W fluorescent light (TL5)) mounted on both sides set at 20-cm from the PBR (Yeh et al., 2010). The light intensity on the PBR was adjusted to ca. 60 lmol/ m2 s. Seed culture of C. vulgaris ESP-31 was inoculated into the reactor with an inoculum size of 20 mg/l. The reactor was operated at 25 °C, pH 6.2, 150 rpm agitation with CO2 aeration (2%, 0.2 vvm). During microalgal growth, the liquid sample was collected from the sealed glass vessel with respect to time to determine microalgal biomass concentration, pH, residual nitrogen concentration and lipid content of the microalgal biomass. The microalga was grown in the PBR on three different media (namely, Basal medium, Modified Bristol’s medium CZ-M1 (Ip and Chen, 2005) and MBL medium). The compositions of these media are given in Table 1. Although the nitrogen source of the three media was the same (i.e., nitrate), the nitrate concentration in Basal, Modified Bristol’s and MBL medium was 0.766, 0.547 and 0.062 g/l, respectively. The nitrate concentrations of the
Table 1 Compositions of Basal medium, Modified Bristol’s medium CZ-M1 and MBL medium. Compositions
Basal medium (g/l)
Modified Bristol’s medium (CZ-M1) (g/l)
MBL medium (g/l)
Medium type
Nitrogen rich Nutrient rich
Nitrogen rich
Nitrogen poor
Nutrient poor
Nutrient poor
0.025 0.025 0.75
0.0368 0.085
NaCl CaCl22H2O NaNO3 KNO3 MgSO47H2O NaHCO3 K2HPO4 KH2PO4 Na2O3Si9H2O FeSO47H2O FeCl36H2O EDTA2Na H3BO3 MnCl24H2O MnSO47H2O ZnSO47H2O Na2MoO42H2O (NH4)6Mo7O247H2O CuSO45H2O Co(NO3)26H2O CoCl26H2O
0.1106 1.25 1
0.075
1.25
0.075 0.175
0.037 0.0126 0.0087 0.0284
0.0498 0.005 0.5 0.1142 0.0144 0.0882 0.0119 0.0157 0.0049
0.000061 0.000169 0.000287 0.00000124 0.0000025
0.00315 0.00436 0.001 0.00018 0.000022 0.000006 0.00001 0.00001
former two are about 10 times higher than that of the latter, thus the MBL medium is considered a nitrogen-poor one, while the other two are considered nitrogen rich. As for the trace metal ions, the three media contain similar trace elements (i.e., Fe, Zn, Mn, Mo, Cu, Co). However, since the concentration of trace metal ions in Basal medium is 10–6000 times higher than those of the other two, Basal medium is considered nutrient rich, while the other two are considered nutrient poor. At the same time, different carbon sources were also used for the growth of C. vulgaris ESP-31. Inorganic carbons (NaHCO3 and CO2) were used as the carbon source in photoautotrophic cultivation, while organic carbon (glucose) was used in heterotrophic and photoheterotrophic cultivation. Mixotrophic cultivation, which means the microalgae could undergo photosynthesis and simultaneously use both organic and inorganic carbon as carbon sources, was also investigated in this study. The effects of these cultivation conditions on microalga growth and lipid production were investigated. 2.3. Determination of microalgal biomass concentration The biomass concentration of the culture in the photobioreactor was monitored regularly by optical density measurement at a wavelength of 688 nm (i.e., OD688) using a spectrophotometer (model U-2001, Hitachi, Tokyo, Japan) after appropriate dilution with deionized water. The OD688 values were converted to dry cell weight (DCW) concentration via appropriate calibration, as indicated below. Under phototrophic and heterotrophic conditions:
Biomass concentration ðg dry cell=lÞ ¼ 0:1999 OD688 ðR2 ¼ 0:9073Þ Under photoheterotrophic and mixotrophic conditions:
Biomass concentration ðg dry cell=lÞ ¼ 0:2917 OD688 ðR2 ¼ 0:9352Þ
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2.7. Determination of protein content
Table 2 Characteristics of different cultivation conditions. Cultivation condition
Energy source
Carbon source
Carbon source used in this study
Phototrophic
Light
CO2 or NaHCO3
Heterotrophic
Organic carbon Light Light and organic carbon
Inorganic carbon Organic carbon Organic carbon Inorganic and organic carbon
Glucose CO2 and glucose
Photoheterotrophic Mixotrophic
Glucose
Total nitrogen content of microalga was detected by an elemental analyzer (Elementar vario EL III). The protein concentration of microalga was estimated by the obtained nitrogen content according to the correlation reported in the literature (i.e., protein concentration = nitrogen content 6.25) (Fuentes et al., 2000). 2.8. Determination of carbohydrate content Total carbohydrate concentration of microalga was determined by the phenol sulfuric method (DuBois et al., 1956).
2.4. Determination of nitrate concentration 3. Results and discussion Nitrate concentration in medium was determined by a colorimetric method (Chiu et al., 2008). Samples were taken from microalga culture and the absorbance (at 220 nm) of the supernatant was measured with a spectrophotometer (model U-2001, Hitachi, Tokyo, Japan) after appropriate dilution with deionized water. The calibration between the absorbance and nitrogen concentration was established using potassium nitrate as the standard. 2.5. Determination of lipid content The lipid content and composition were determined as fatty acid methyl esters (FAMEs) through the direct transesterification method (Chen et al., 2010; Su et al., 2007; Yeh et al., 2010). The biomass of microalgae was harvested by centrifugation (5000 rpm), and then washed twice with deionized water to remove the salt in the medium. The collected biomass was dried by lyophilization. A fixed amount (0.04 g) of the lyophilized cells were mixed with 8 ml of 0.5 N KOH in ethanol and disrupted by bead-beater (MM400, Retsch, Germany) for 25 min. The mixture was heated to 100 °C for 15 min for saponification and then cooled to room temperature. For esterification, 8 ml of 0.7 N HCl in methanol and 14% (v/v) BF3/CH3OH (Sigma–Aldrich, USA) were added to the mixture and heated to 100 °C for 15 min. After cooling to room temperature, 2 ml of saturated NaCl solution was added for preventing emulsification. The FAMEs formed due to transesterification were extracted by n-hexane. The external standard (methyl pentadecanoate (C15:0), Sigma) was added after transesterification to determined the lipid content. The composition of fatty acid methyl esters after direct transesterification was analyzed using a gas chromatograph (GC-2014, Shimadzu, Kyoto, Japan) equipped with a flame ionization detector (FID). Samples were injected into a 100 m-long capillary column (SP™2560, Supelco, Bellefonte, PA, USA) with an internal diameter of 0.25 mm. Helium was used as the carrier gas with a flow rate of 20 cm/s. The temperature of the injector and detector were both set at 260 °C. The oven temperature was initially set at 140 °C for 5 min, and increased from 140 to 240 °C at an increasing rate of 4 °C/min, and finally held at 240 °C for 20 min. 2.6. Determination of lipid productivity The liquid productivity (Plipid) was determined based on the calculation indicated in Eq. (1):
Plipids ðg=l=dÞ ¼
3.1. The effects of media and cultivation conditions on biomass production and composition of C. vulgaris ESP-31 It is known that the growth rate and the biomass composition of microalgae would vary under different medium composition and cultivation conditions (Chen et al., 2011). To determine a suitable medium for the growth and lipid production of C. vulgaris ESP31, three kinds of media of different richness with regard to nitrogen source and nutrient (mainly trace metal ions) content (Table 1) were used to cultivate the microalga in this study, because lipid accumulation in microalgae is often triggered under nitrogen or nutrient limiting conditions (Illman et al., 2000; Li et al., 2008; Solovchenko et al., 2008; Yeesang and Cheirsilp, 2011; Yeh and Chang, 2011). On the other hand, Chlorella sp. can adapt to different growth conditions such as phototrophic, heterotrophic and mixotrophic cultivation (Chen et al., 2011; Cheng et al., 2009; Chiu et al., 2008; Liang et al., 2009; Xiong et al., 2008), and some reports have indicated that the lipid content and productivity could be greatly dependent on those conditions. In general, microalgae could be grown under four different cultivation conditions (Chen et al., 2011; Chojnacka and Marquez-Rocha, 2004; Mata et al., 2010), as described in Table 2. In this work, all four conditions shown in Table 2 were used to grow C. vulgaris ESP-31 to examine which results in the highest biomass and lipid productivity. Fig. 1 shows the of biomass production performance of C. vulgaris ESP-31 grown under different combinations of media (Basal medium, Modified Bristol’s medium, and MBL medium) and cultivation conditions (phototrophic, heterotrophic, mixotrophic and photoheterotrophic cultivation). The inter-correlation of cell growth with medium and cultivation conditions depicted in Fig. 1 shows a biomass concentration of below 0.5 g/l was observed under phototrophic (using 1 g/l NaHCO3 as carbon source) and heterotrophic (using 10 g/l glucose as carbon source) growth. The use of a higher NaHCO3 concentration (1.2–1.6 g/l) for phototrophic cultivation resulted in high pH (10 and 11) in the broth, thereby causing inhibitory effects with regard to the microalgae growth (Yeh et al., 2010). This limited the biomass production to no more than 0.46 g/l when using NaHCO3-amended Basal medium for microalgal growth (Fig. 1). In contrast, when CO2 was used as the carbon source for phototrophic growth on Basal medium, the highest biomass production reached 5.00 g/l (Fig. 1). In phototrophic cultivation, better cell growth was obtained when using a nitrogen-rich medium (i.e., Basal medium and Modified Bristol’s medium) when
Cumulative microalgae biomass production ðgÞ lipid content ð%Þ Working volume ðlÞ cultivation time ðdÞ
ð1Þ
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Fig. 1. Biomass production performance of C. vulgaris ESP-31 grown on different media (Basal, Modified Bristol’s and MBL medium) under different cultivation conditions (phototrophic, heterotrophic, mixotrophic and photoheterotrophic cultivation).
compared with growth on nitrogen-poor MBL medium (Fig. 1), indicating that nitrogen source concentration is essential in supporting photoautotrophic growth of the microalgal strain. C. vulgaris ESP-31 cannot grow well under heterotrophic conditions. However, with a light supply, the microalga grew efficiently, as the biomass concentration in photoheterotrophic culture was 15 times higher than that obtained under heterotrophic cultivation on Basal medium and Modified Bristol’s medium (Fig. 1). Mixotrophic growth was also effective, with similar biomass production efficiency to that seen with photoheterotrophic growth (Fig. 1). Apparently, light energy is required for the C. vulgaris ESP-31 strain to efficiently assimilate organic carbon sources. Some C. vulgaris strains seem to display better growth under photoheterotrophic conditions. For instance, Liang et al. (2009) reported that when their C. vulgaris strain was grown on 1% glucose with light (photoheterotrophic condition), the highest biomass production (2 g/l) and lipid productivity (54.0 mg/l/d) achieved were superior to those obtained under heterotrophic conditions with the same glucose concentration. These results are quite similar to what was observed with the C. vulgaris ESP-31 strain. However, there are also some microalgal species that can grow very fast on organic carbon sources in the dark (Cheng et al., 2009; Liang et al., 2009; Xiong et al., 2008), and the glucose metabolism is different under dark and light conditions. Pentose Phosphate Pathway (PPP) is the main pathway under darkness, while under light it is the Embden-Meyerhof Pathway (EMP) (Bashan et al., 2011). Some microalgae species cannot metabolize glucose under anaerobic-dark conditions due to the low levels of the lactate dehydrogenase enzyme; however, there are some species that still cannot assimilate glucose even after having the enzymes (Bashan et al., 2011; Neilson and Lewin, 1974). Photoheterotrophic organisms can only grow when both light and glucose are present, implying that glucose is not the energy source but rather the building material. In photoheterotrophic cultivation, light energy is used as the energy source, which is transformed into chemical energy, such as ATP and NADPH (Chojnacka and Marquez-Rocha, 2004). Our results show that the C. vulgaris ESP-31 strain can use both inorganic and organic carbon sources for growth, but light is necessary in both situations. The results also show that the biomass production is not only
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dependent on growth conditions, but also on the medium used. Therefore, both factors should be taken into account simultaneously to optimize microalgal growth. The main components of microalgae are lipid, protein and carbohydrate. Under common condition, the lipid, protein and carbohydrate content of C. vulgaris is about 14–22%, 51–58%, and 12– 17%, respectively (Spolaore et al., 2006). Fig. 2 shows the biomass composition of C. vulgaris ESP-31 under different growth conditions. Carbohydrate content was about 30–40% in most cultivation conditions with CO2 aeration (phototrophic and mixotrophic condition), while a higher carbohydrate content (about 50–60%) was obtained when using glucose as the only carbon source (heterotrophic and photoheterotrophic condition). Fig. 2 also shows that under all cultivation conditions cell growth on Basal medium (rich in nitrogen and nutrient content) led to lower lipid content (5–22%), while higher lipid content (19–53%) occurred in MBL medium, which has lower nitrogen and nutrient content. The highest lipid content (53%) was obtained when using MBL medium under phototrophic growth. This clearly shows that, as often mentioned in the literature, a deficiency in nitrogen source or nutrients could improve lipid and carbohydrate accumulation (Griffiths and Harrison, 2009; Mata et al., 2010; Yeh and Chang, 2011). The high lipid and carbohydrate content observed in C. vulgaris ESP-31 might due to the cells were collected at stationary phase of the growth. In the exponential growth phase of microalgae, the biomass contains mostly proteins (Mata et al., 2010). However, the nitrogen and nutrient usually deplete at the stationary phase and the lipid/carbohydrate start to accumulate in microalgal cells. Thus, the medium composition and growth conditions are the most critical factor affecting the lipid and carbohydrate content of the microalgae. Furthermore, there is a common trend that higher lipid and carbohydrate content always appears with lower protein content (Fig. 2). This result seems reasonable, since lipid and carbohydrate accumulation usually occurs under nitrogen limiting condition, and so the intracellular proteins may be degraded to supply nitrogen to maintain the metabolic functions in microalgae (Gouveia et al., 2009; Mata et al., 2010). 3.2. The effects of media composition and cultivation conditions on lipid productivity of C. vulgaris ESP-31 According to the biomass composition data (Fig. 2), C. vulgaris ESP-31 has the potential for lipid production, as its lipid content could reach up to 53% under suitable medium and cultivation conditions. Although the results indicate that the use of MBL medium could lead to higher lipid contents, the microalgal growth rate was very low on MBL medium due to its low nitrogen and nutrient content. Therefore, from an engineering perspective, it would be more important and practical to evaluate the lipid production performance by simultaneously considering lipid content and biomass production rate, rather than simply emphasizes the lipid content (Griffiths and Harrison, 2009; Yeh and Chang, 2011). As a result, in this study, lipid productivity (Plipid g/ l/d), which combines the dual effects of lipid content and biomass productivity, was used as the performance index to examine the efficiency of lipid production from the microalgal strain (Chen et al., 2011; Griffiths and Harrison, 2009; Yeh and Chang, 2011). Table 3 compares the lipid productivities obtained under different media and cultivation conditions. Regardless of the media used, the two unfavorable cultivation conditions (i.e., phototrophic culture with NaHCO3 and heterotrophic culture) resulted in very poor lipid productivity (2.7–7.7 mg/l/d) (Table 3). For the rest of the cultivation conditions, the lipid productivity obtained from using Basal medium only varied slightly (56–69 mg/l/d), but for the microalga grown on MBL medium and Modified Bristol’s
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Fig. 2. Biomass composition of C. vulgaris ESP-31 grown under different cultivation conditions (phototrophic, heterotrophic, mixotrophic and photoheterotrophic cultivation) on (a) Basal medium, (b) Modified Bristol’s medium and (c) MBL medium.
medium, the lipid productivity varied significantly (21–105 mg/l/d and 40–144 mg/l/d, respectively) when different cultivation conditions were used (Table 3). Higher lipid productivities were obtained when using glucose as the carbon source in photoheterotrophic or mixotrophic cultures (Table 3). Although the highest lipid content (53%) was obtained under phototrophic growth using CO2 as the carbon source on MBL medium (Figs. 2 and 3), the
highest lipid productivity (143.9 mg/l/d) occurred in the mixotrophic culture on Modified Bristol’s medium (Table 3). This suggests that the best lipid production performance may not necessarily originate from the microalgal biomass with the highest lipid content, whereas the cell growth rate and biomass productivity may play a determining role in the productivity of microalgal lipid. The highest productivity (143.9 mg/l/d) obtained from this study is also among the highest that has yet been reported in the literature (Chen et al., 2011; Mata et al., 2010). Fig. 3 shows the time-course profiles of biomass production, residual nitrogen concentration and lipid content under different media and cultivation conditions. The specific growth rate and nitrogen utilization rate under photoheterotrophic conditions are much higher than under phototrophic ones (on Basal and Modified Bristol’s medium), resulting in higher lipid productivity in photoheterotrophic cultures even though the lipid contents are similar in both conditions (Fig. 3). Moreover, the use of mixotrophic conditions with all medium could obtain the highest nitrogen utilization rate, lipid accumulation rate, lipid content (40–53%) and lipid productivity (67–144 mg/l/d). In all cases, the microalgal lipids were significantly accumulated after the nitrogen deficiency, especially in mixotrophic conditions (Fig. 3). Darzins et al. (2008) examined the fatty acid synthesis pathway in chloroplasts, and mentioned that the conversion of acetyl CoA to malonyl CoA is the key step in fatty acid synthesis. The formation of malonyl CoA from acetyl CoA and CO2 (from HCO 3 ) is catalyzed by acetyl CoA carboxylase (ACCase) (Darzins et al., 2008; Ohlrogge and Browse, 1995). Therefore, when the microalgae assimilate glucose as the carbon source, the culture enters the nitrogen starvation period faster and, with the addition of CO2 under mixotrophic conditions, the rate of lipid accumulation would be stimulated. This may explain why mixotrophic cultures resulted in higher lipid productivities with the microalga. Although using glucose for microalgal growth resulted in greater lipid productivity, the conversion of CO2 to microalgal lipid is more promising from the perspective of CO2 mitigation (Chisti, 2007; Mata et al., 2010). The lipid contents of phototrophic cultures on Basal and Modified Bristol’s media only slightly increased from 18% to 23–27% after nitrogen starvation, while it increased rapidly from 38% to 68% in MBL medium (Fig. 3). This may be because the nitrogen source concentration of MBL medium is much lower than that of the other two media, indicating that the initial nitrogen source concentration might be the key factor influencing the lipid accumulation (Yeh and Chang, 2011). This microalga strain thus still has the potential to produce microalgal lipids for making biodiesel via phototrophic cultivation if an appropriate medium and nitrogen source concentration is used. 3.3. Fatty acid profile of the produced microalgal lipids The performance of microalgal lipid production of C. vulgaris ESP-31 has been described in the earlier sections. It is also equally
Table 3 Lipid productivity of C. vulgaris ESP-31 grown on different media (Basal, Modified Bristol’s and MBL medium) under different cultivation conditions (phototrophic, heterotrophic, photoheterotrophic and mixotrophic cultivation). Medium
Basal Modified Bristol’s MBL
Lipid productivity (mg/l/d) Phototrophic culture (NaHCO3)
Phototrophic culture (CO2)
Heterotrophic culture (glucose)
Photoheterotrophic culture (glucose)
Mixotrophic culture (glucose and CO2)
2.7 ± 0.5 3.7 ± 0.4
56.2 ± 2.9 40.2 ± 0.8
3.5 ± 0.5 3.5 ± 0.1
69.0 ± 2.3 115.4 ± 24.7
67.4 ± 2.5 143.9 ± 0.8
6.3 ± 0.2
51.2 ± 5.5
7.7 ± 0.4
20.7 ± 3.9
104.9 ± 7.2
Data shown are averages of two runs ± SD (standard deviation).
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6
Biomass production (g/L)
Phototrophic (CO2)
Basal medium
Modified Bristol's medium
MBL medium
Photoheterotrophic Mixotrophic
5
4
3
2
1
Nitrate concentration (mg/L)
0 800
600
400
200
Lipid content (wt. %/biomass)
0 70 60 50 40 30 20 10 0 0
4
8
12
16
20
0
4
Time (d)
8
12
Time (d)
16
20
0
4
8
12
16
20
Time (d)
Fig. 3. Time-course profile of biomass production, nitrogen concentration and lipid content of C. vulgaris ESP-31 grown on Basal medium, Modified Bristol’s medium and MBL medium under different cultivation conditions. Data shown are the average of two runs ± SD (standard deviation).
important to determine the quality of the microalgal lipids thus produced to assess the feasibility of using them for biodiesel synthesis. The raw materials used for making biodiesel need to meet some standard regulations issued by local governments. For instance, the ASTM Biodiesel Standard D6751 is executed in the United States, while in European Union vehicles using biodiesel need to follow regulations based on the Standard EN 14214 (Chisti, 2007). The European biodiesel standards have a restriction limiting the FAMEs content with four and more double bonds to 1 mol%, and the linolenic acid methyl ester (C18:3) content to 12 mol% (Chisti, 2007). In addition, lipids with fatty acids containing 16–18 carbon atoms are considered suitable for producing biodiesel (Huang et al., 2010). Therefore, analyzing the fatty acid profile of microalgal lipids produced from C. vulgaris ESP-31 is necessary to assess whether the quality is suitable for biodiesel synthesis (Chisti, 2007; Huang et al., 2010). Table 4 shows the profiles of lipid content and fatty acid composition of C. vulgaris ESP-31 under different medium and cultivation conditions. The main fatty acids in C. vulgaris ESP-31 were palmitic
acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3). Among those, palmitic acid and oleic acid were the two most predominant fatty acid components, accounting for 50–62% of total fatty acids present in the microalgal lipids (Table 4). This lipid profile is in accord with that reported in the literature, indicating that the main fatty acids present in the lipid of Chlorella sp. are normally short chain fatty acids (C14–C18) (Huang et al., 2010). There is a clear trend that for microalgal cells with over 40% lipid content, higher C16:0 and C18:1 content was observed in the microalgal lipids, while for the microalgal lipids with the content under 30%, more C16:0 and C18:2 fatty acids appeared (Table 4). This trend can also be clearly observed in Fig. 4. The relative content of C16:0 increased from 18% to 31% and an increase from 3% to 33% was observed in fatty acid C18:1 when lipid content of microalgae increased from 13% to 63%, whereas the relative content of C18:2 reduced from 26% to 16% for microalgal lipid with the same lipid content range. Similar results were reported by other researchers (Darzins et al., 2008), as they found that in green algae the proportion of
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Table 4 The fatty acids composition of C. vulgaris ESP-31 grown in different media (Basal, Modified Bristol’s and MBL medium) and different cultivation conditions (phototrophic, mixotrophic and photoheterotrophic cultivation). Medium
Cultivation condition
Basal
Phototrophic (CO2)
56.2 ± 2.9
Basal
Photoheterotrophic
69.0 ± 2.3
Basal
Mixotrophic
67.4 ± 2.5
Modified Bristol
Phototrophic (CO2)
40.2 ± 0.8
Modified Bristol
Photoheterotrophic
115.4 ± 24.7
Modified Bristol
Mixotrophic
143.9 ± 0.8
MBL
Phototrophic (CO2)
51.2 ± 5.5
MBL
Photoheterotrophic
20.7 ± 3.9
MBL
Mixotrophic
Fatty acid content (wt.%/dry weight of biomass)a or relative content (%)b
Lipid productivity (mg/l/d)
104.9 ± 7.2
Total
C16:0
C16:1
C18:0
C18:1
C18:2
C18:3
Others
23.89a 100b 13.14a 100b 45.44a 100b
5.24 21.93 2.16 16.44 13.09 28.81
0.06 0.25 0.28 2.13 0.57 1.25
2.39 10.00 1.96 14.92 3.03 6.67
3.31 13.86 0.48 3.65 11.29 24.85
6.16 25.78 3.31 25.19 9.36 20.60
1.18 4.94 0.97 7.38 3.08 6.78
5.55 23.23 3.98 30.29 5.02 11.05
21.72a 100b 30.18a 100b 57.61a 100b
3.89 17.91 6.87 22.76 16.59 28.80
0.04 0.18 0.23 0.76 0.38 0.66
2.38 10.96 2.38 7.89 3.58 6.21
1.23 5.66 8.06 26.71 18.94 32.88
5.03 23.16 7.06 23.39 9.29 16.13
1.98 9.12 1.03 3.41 2.79 4.84
7.17 33.01 4.55 15.08 6.04 10.48
62.95a 100b 40.62a 100b 49.92a 100b
19.71 31.31 10.75 26.46 14.63 29.31
0.06 0.10 0.27 0.66 0.50 1.00
4.44 7.05 2.87 7.07 3.83 7.67
18.48 29.36 10.43 25.68 14.63 29.31
10.04 15.95 7.26 17.87 8.17 16.37
3.67 5.83 3.60 8.86 3.15 6.31
6.55 10.41 5.44 13.39 5.01 10.04
produced by C. vulgaris ESP-31 contained over 60–68% of saturated and monounsaturated fatty acids, thereby being suitable for applications in the biodiesel industry.
Relative content of fatty acid (%)
35 30
Acknowledgements
25
The authors gratefully acknowledge financial support from Taiwan’s National Science Council under grant numbers NSC 1003113-E-006-016- and NSC 99-3113-P-110-001-. The support from the Top University grant of National Cheng Kung University is also appreciated.
20 15 10
C16:0 C18:1 C18:2
5 0
0
10
20
30
40
50
60
References
70
Lipid content (wt. % per biomass) Fig. 4. The relative content of fatty acids (C16:0, C18:1 and C18:2) at different lipid content of C. vulgaris ESP-31 grown in different media (Basal, Modified Bristol’s and MBL medium) and different cultivation conditions (phototrophic, mixotrophic and photoheterotrophic cultivation).
polyunsaturated C18:2 fatty acid in the microalgal biomass tends to decrease after nitrogen starvation. In this study, the lipids produced consisted of over 60–68% of saturated and monounsaturated fatty acids (palmitic acid (C16:0), stearic acid (C18:0) and oleic acid (C18:1)), which are considered suitable for synthesizing biodiesel (Sheehan et al., 1998). Hence, the C. vulgaris ESP-31 strain seems to be an excellent candidate as biodiesel feedstock. 4. Conclusions This study demonstrates the feasibility of using an indigenous microalga C. vulgaris ESP-31 as feedstock for biodiesel production. The cell growth and lipid accumulation are greatly affected by medium composition and cultivation conditions. Higher biomass production (2–5 g/l) was obtained using nitrogen-rich medium under various growth conditions, whereas higher lipid content (20–53%) was achieved by using a nitrogen-limiting MBL medium. The highest lipid productivity (144 mg/l/d) was obtained from mixotrophic cultivation on Modified Bristol’s medium. The lipid
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K.-L. Yeh, J.-S. Chang / Bioresource Technology 105 (2012) 120–127 Ho, S.H., Chen, C.Y., Yeh, K.L., Chen, W.M., Lin, C.Y., Chang, J.S., 2010. Characterization of photosynthetic carbon dioxide fixation ability of indigenous Scenedesmus obliquus isolates. Biochem. Eng. J. 53, 57–62. 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. Huang, G.H., Chen, F., Wei, D., Zhang, X.W., Chen, G., 2010. Biodiesel production by microalgal biotechnology. Appl. Energy 87, 38–46. Illman, A.M., Scragg, A.H., Shales, S.W., 2000. Increase in Chlorella strains calorific values when grown in low nitrogen medium. Enzyme Microb. Technol. 27, 631– 635. Ip, P.F., Chen, F., 2005. Production of astaxanthin by the green microalga Chlorella zofingiensis in the dark. Process Biochem. 40, 733–738. Li, Q., Du, W., Liu, D.H., 2008. Perspectives of microbial oils for biodiesel production. Appl. Microbiol. Biotechnol. 80, 749–756. Li, X.F., Xu, H., Wu, Q.Y., 2007. Large-scale biodiesel production from microalga Chlorella protothecoides through heterotropic cultivation in bioreactors. Biotechnol. Bioeng. 98, 764–771. Liang, Y.N., Sarkany, N., Cui, Y., 2009. Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions. Biotechnol. Lett. 31, 1043–1049. Mata, T.M., Martins, A.A., Caetano, N.S., 2010. Microalgae for biodiesel production and other applications: a review. Renew. Sustainable Energy Rev. 14, 217–232. Neilson, A.H., Lewin, R.A., 1974. The uptake and utilization of organic carbon by algae: an essay in comparative biochemistry⁄. Phycologia 13, 227–264. Ohlrogge, J., Browse, J., 1995. Lipid biosynthesis. Plant Cell 7, 957–970. Posten, C., Schaub, G., 2009. Microalgae and terrestrial biomass as source for fuels-A process view. J. Biotechnol. 142, 64–69.
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Sheehan, J., Dunahay, T., Benemann, J., Roessler, P., 1998. A Look Back at the US Department of Energy’s Aquatic Species Program: Biodiesel from Algae. National Renewable Energy Laboratory, USA. Shi, X.M., Chen, F., Yuan, J.P., Chen, H., 1997. Heterotrophic production of lutein by selected Chlorella strains. J. Appl. Phycol. 9, 445–450. Solovchenko, A.E., Khozin-Goldberg, I., Didi-Cohen, S., Cohen, Z., Merzlyak, M.N., 2008. Effects of light intensity and nitrogen starvation on growth, total fatty acids and arachidonic acid in the green microalga Parietochloris incisa. J. Appl. Phycol. 20, 245–251. Spolaore, P., Joannis-Cassan, C., Duran, E., Isambert, A., 2006. Commercial applications of microalgae. J. Biosci. Bioeng. 101, 87–96. Su, C.H., Giridhar, R., Chen, C.W., Wu, W.T., 2007. A novel approach for medium formulation for growth of a microalga using motile intensity. Bioresour. Technol. 98, 3012–3016. Xiong, W., Li, X.F., Xiang, J.Y., Wu, Q.Y., 2008. High-density fermentation of microalga Chlorella protothecoides in bioreactor for microbio-diesel production. Appl. Microbiol. Biotechnol. 78, 29–36. Yeesang, C., Cheirsilp, B., 2011. Effect of nitrogen, salt, and iron content in the growth medium and light intensity on lipid production by microalgae isolated from freshwater sources in Thailand. Bioresour. Technol. 102, 3034–3040. Yeh, K.-L., Chang, J.-S., 2011. Nitrogen starvation strategies and photobioreactor design for enhancing lipid content and lipid production of a newly isolated microalga Chlorella vulgaris ESP-31: implications for biofuels. Biotechnol. J. doi:10.1002/biot.201000433. Yeh, K.L., Chang, J.S., Chen, W.M., 2010. Effect of light supply and carbon source on cell growth and cellular composition of a newly isolated microalga Chlorella vulgaris ESP-31. Eng. Life Sci. 10, 201–208.
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Effects of cultivation conditions and media composition on cell growth and lipid productivity of indigenous microalga Chlorella vulgaris ESP-31 Kuei-Ling Yeh a, Jo-Shu Chang a,b,c,⇑ a
Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan University Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan c Research Center for Energy Technology and Strategy, National Cheng Kung University, Tainan, Taiwan b
a r t i c l e
i n f o
Article history: Received 1 October 2011 Received in revised form 23 November 2011 Accepted 26 November 2011 Available online 2 December 2011 Keywords: Chlorella vulgaris Lipid productivity Microalgal lipid Medium composition Cultivation condition
a b s t r a c t The growth and lipid productivity of an isolated microalga Chlorella vulgaris ESP-31 were investigated under different media and cultivation conditions, including phototrophic growth (NaHCO3 or CO2, with light), heterotrophic growth (glucose, without light), photoheterotrophic growth (glucose, with light) and mixotrophic growth (glucose and CO2, with light). C. vulgaris ESP-31 preferred to grow under phototrophic (CO2), photoheterotrophic and mixotrophic conditions on nitrogen-rich medium (i.e., Basal medium and Modified Bristol’s medium), reaching a biomass concentration of 2–5 g/l. The growth on nitrogen-limiting MBL medium resulted in higher lipid accumulation (20–53%) but slower growth rate. Higher lipid content (40–53%) and higher lipid productivity (67–144 mg/l/d) were obtained under mixotrophic cultivation with all the culture media used. The fatty acid composition of the microalgal lipid comprises over 60–68% of saturated fatty acids (i.e., palmitic acid (C16:0), stearic acid (C18:0)) and monounsaturated acids (i.e., oleic acid (C18:1)). This lipid composition is suitable for biodiesel production. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Microalgal biomass, containing lipids, starch, cellulose, proteins, and so on, is considered a promising feedstock for producing a variety of renewable fuels, such as biodiesel, bioethanol, biohydrogen and methane (Carioca, 2010; Chisti, 2008; Posten and Schaub, 2009). Microalgal lipids have attracted much attention as future raw materials for biodiesel synthesis due to the potential of attaining much higher lipid productivity than is possible with other lipidbased energy crops (Chisti, 2007; Griffiths and Harrison, 2009). Several strategies have been applied to improve microalgae growth and lipid content. These include optimization of the medium compositions (e.g., type of carbon source, vitamins, salts and nutrients), physical parameters (e.g., pH, temperature and light intensity), and type of metabolism (e.g., phototrophic, heterotrophic, mixotrophic and photoheterotrophic growth) (Chojnacka and Marquez-Rocha, 2004; Mata et al., 2010). In particular, the type of cultivation method, using different energy sources (light or organic) and carbon sources (inorganic or organic), is always recognized as a key factor that significantly influences the growth and lipid accumulation of microalgae (Chen et al., 2011; Mata et al., 2010). ⇑ Corresponding author at: Department of Chemical Engineering, National Cheng Kung University, Tainan 710, Taiwan. Tel.: +886 6 2757575x62651; fax: +886 6 2357146. E-mail address: [email protected] (J.-S. Chang). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.11.103
Photoheterotrophic conditions are rarely mentioned in the literature, and it is usually confusing to distinguish mixotrophic from photoheterotrophic growth (Griffiths and Harrison, 2009; Mata et al., 2010). In photoheterotrophic cultivation, the microalgae require light as an energy source while using organic materials as the carbon source. In contrast, both organic carbon and CO2 are essential carbon sources in mixotrophic growth, as a light source is also supplied (Chojnacka and Marquez-Rocha, 2004). Although photoautotrophic conditions are commonly used in microalgae cultivation, some reports noted that higher biomass and lipid productivity of Chlorella sp. could be obtained under heterotrophic and mixotrophic conditions (Chen et al., 2011; Cheng et al., 2009; Li et al., 2007; Liang et al., 2009; Xiong et al., 2008). The nitrogen source and concentration in the medium used are also known two of the most crucial factors affecting the lipid content of microalgae (Hsieh and Wu, 2009; Yeh and Chang, 2011). Therefore, it is of importance to utilize appropriate cultivation conditions and medium composition (especially nitrogen source and concentration) to achieve the best lipid production performance of microalgae species. In this study, an indigenous microalgal isolate (identified as Chlorella vulgaris ESP-31) was examined with regard to its effectiveness for biomass and lipid production under different medium and cultivation conditions. To identify the lipid production performance of C. vulgaris ESP-31, the lipid content and lipid productivity were monitored with respect to time when the
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microalga was grown under different medium and cultivation conditions. The fatty acid profile of the microalgal lipid was also determined to evaluate the feasibility of using the produced microalgal lipid for biodiesel synthesis. This study aimed to identify the best conditions for the cell growth and lipid accumulation of C. vulgaris ESP-31, and to evaluate the potential of using the microalgal biomass thus produced as biodiesel feedstock. 2. Methods 2.1. Microalga strain and culture medium C. vulgaris ESP-31 was isolated from a shrimp-culturing pond located in South Taiwan. The freshwater sample collected from Southern Taiwan was inoculated into BG11 medium and grown at 25 °C in an artificial light cabinet under illumination with fluorescent lamps (60 lmol/m2 s, light/dark cycle: 16/8 h). The culture was then spread on BG11 medium agar plates and the appeared colonies were subcultured into the same medium until the pure strain was obtained (Ho et al., 2010). The identity of the microalgal isolate was determined through plastid 23S rRNA gene sequence analysis. The genomic DNA of the microalga was extracted using the Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA). The plastid 23S rRNA gene was amplified by polymerase chain reaction (PCR) using two universal algal primers reported previously (Ho et al., 2010). The sequence analysis was conducted using a DNA sequencer (ABI Prism 310; Applied Biosystems) and the DNA sequences were assembled using the Fragment Assembly System program from the Wisconsin package version 9.1. The sequences of the microalgal strains were compared against plastid 23S rRNA gene sequences available from the GenBank databases. Multiple sequence alignment including microalgal strains and their closest relatives was performed using BioEdit software and MEGA4 (Ho et al., 2010). The medium used for the preculture of C. vulgaris ESP-31 was Basal medium (Shi et al., 1997) that consisted of (g/l): KNO3, 1.25; KH2PO4, 1.25; MgSO47H2O, 1; CaCl2, 0.0835; H3BO3, 0.1142; FeSO47H2O, 0.0498; ZnSO47H2O, 0.0882; MnCl24H2O, 0.0144; MoO3, 0.0071; CuSO45H2O, 0.0157; Co(NO3)26H2O, 0.0049; EDTA2Na, 0.5. The microalga was regularly grown at 25 °C for 4–5 days with a continuous supply of 2% CO2 at an aeration rate of 0.2 vvm. The microalga culture was illuminated all day with a light intensity of ca. 60 lmol/m2 s. 2.2. Operation of photobioreactor and culture conditions The photobioreactor (PBR) used to cultivate C. vulgaris ESP-31 was a 1-liter glass vessel (15.5 cm in length and 9.5 cm in diameter) equipped with an external light source (14 W fluorescent light (TL5)) mounted on both sides set at 20-cm from the PBR (Yeh et al., 2010). The light intensity on the PBR was adjusted to ca. 60 lmol/ m2 s. Seed culture of C. vulgaris ESP-31 was inoculated into the reactor with an inoculum size of 20 mg/l. The reactor was operated at 25 °C, pH 6.2, 150 rpm agitation with CO2 aeration (2%, 0.2 vvm). During microalgal growth, the liquid sample was collected from the sealed glass vessel with respect to time to determine microalgal biomass concentration, pH, residual nitrogen concentration and lipid content of the microalgal biomass. The microalga was grown in the PBR on three different media (namely, Basal medium, Modified Bristol’s medium CZ-M1 (Ip and Chen, 2005) and MBL medium). The compositions of these media are given in Table 1. Although the nitrogen source of the three media was the same (i.e., nitrate), the nitrate concentration in Basal, Modified Bristol’s and MBL medium was 0.766, 0.547 and 0.062 g/l, respectively. The nitrate concentrations of the
Table 1 Compositions of Basal medium, Modified Bristol’s medium CZ-M1 and MBL medium. Compositions
Basal medium (g/l)
Modified Bristol’s medium (CZ-M1) (g/l)
MBL medium (g/l)
Medium type
Nitrogen rich Nutrient rich
Nitrogen rich
Nitrogen poor
Nutrient poor
Nutrient poor
0.025 0.025 0.75
0.0368 0.085
NaCl CaCl22H2O NaNO3 KNO3 MgSO47H2O NaHCO3 K2HPO4 KH2PO4 Na2O3Si9H2O FeSO47H2O FeCl36H2O EDTA2Na H3BO3 MnCl24H2O MnSO47H2O ZnSO47H2O Na2MoO42H2O (NH4)6Mo7O247H2O CuSO45H2O Co(NO3)26H2O CoCl26H2O
0.1106 1.25 1
0.075
1.25
0.075 0.175
0.037 0.0126 0.0087 0.0284
0.0498 0.005 0.5 0.1142 0.0144 0.0882 0.0119 0.0157 0.0049
0.000061 0.000169 0.000287 0.00000124 0.0000025
0.00315 0.00436 0.001 0.00018 0.000022 0.000006 0.00001 0.00001
former two are about 10 times higher than that of the latter, thus the MBL medium is considered a nitrogen-poor one, while the other two are considered nitrogen rich. As for the trace metal ions, the three media contain similar trace elements (i.e., Fe, Zn, Mn, Mo, Cu, Co). However, since the concentration of trace metal ions in Basal medium is 10–6000 times higher than those of the other two, Basal medium is considered nutrient rich, while the other two are considered nutrient poor. At the same time, different carbon sources were also used for the growth of C. vulgaris ESP-31. Inorganic carbons (NaHCO3 and CO2) were used as the carbon source in photoautotrophic cultivation, while organic carbon (glucose) was used in heterotrophic and photoheterotrophic cultivation. Mixotrophic cultivation, which means the microalgae could undergo photosynthesis and simultaneously use both organic and inorganic carbon as carbon sources, was also investigated in this study. The effects of these cultivation conditions on microalga growth and lipid production were investigated. 2.3. Determination of microalgal biomass concentration The biomass concentration of the culture in the photobioreactor was monitored regularly by optical density measurement at a wavelength of 688 nm (i.e., OD688) using a spectrophotometer (model U-2001, Hitachi, Tokyo, Japan) after appropriate dilution with deionized water. The OD688 values were converted to dry cell weight (DCW) concentration via appropriate calibration, as indicated below. Under phototrophic and heterotrophic conditions:
Biomass concentration ðg dry cell=lÞ ¼ 0:1999 OD688 ðR2 ¼ 0:9073Þ Under photoheterotrophic and mixotrophic conditions:
Biomass concentration ðg dry cell=lÞ ¼ 0:2917 OD688 ðR2 ¼ 0:9352Þ
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2.7. Determination of protein content
Table 2 Characteristics of different cultivation conditions. Cultivation condition
Energy source
Carbon source
Carbon source used in this study
Phototrophic
Light
CO2 or NaHCO3
Heterotrophic
Organic carbon Light Light and organic carbon
Inorganic carbon Organic carbon Organic carbon Inorganic and organic carbon
Glucose CO2 and glucose
Photoheterotrophic Mixotrophic
Glucose
Total nitrogen content of microalga was detected by an elemental analyzer (Elementar vario EL III). The protein concentration of microalga was estimated by the obtained nitrogen content according to the correlation reported in the literature (i.e., protein concentration = nitrogen content 6.25) (Fuentes et al., 2000). 2.8. Determination of carbohydrate content Total carbohydrate concentration of microalga was determined by the phenol sulfuric method (DuBois et al., 1956).
2.4. Determination of nitrate concentration 3. Results and discussion Nitrate concentration in medium was determined by a colorimetric method (Chiu et al., 2008). Samples were taken from microalga culture and the absorbance (at 220 nm) of the supernatant was measured with a spectrophotometer (model U-2001, Hitachi, Tokyo, Japan) after appropriate dilution with deionized water. The calibration between the absorbance and nitrogen concentration was established using potassium nitrate as the standard. 2.5. Determination of lipid content The lipid content and composition were determined as fatty acid methyl esters (FAMEs) through the direct transesterification method (Chen et al., 2010; Su et al., 2007; Yeh et al., 2010). The biomass of microalgae was harvested by centrifugation (5000 rpm), and then washed twice with deionized water to remove the salt in the medium. The collected biomass was dried by lyophilization. A fixed amount (0.04 g) of the lyophilized cells were mixed with 8 ml of 0.5 N KOH in ethanol and disrupted by bead-beater (MM400, Retsch, Germany) for 25 min. The mixture was heated to 100 °C for 15 min for saponification and then cooled to room temperature. For esterification, 8 ml of 0.7 N HCl in methanol and 14% (v/v) BF3/CH3OH (Sigma–Aldrich, USA) were added to the mixture and heated to 100 °C for 15 min. After cooling to room temperature, 2 ml of saturated NaCl solution was added for preventing emulsification. The FAMEs formed due to transesterification were extracted by n-hexane. The external standard (methyl pentadecanoate (C15:0), Sigma) was added after transesterification to determined the lipid content. The composition of fatty acid methyl esters after direct transesterification was analyzed using a gas chromatograph (GC-2014, Shimadzu, Kyoto, Japan) equipped with a flame ionization detector (FID). Samples were injected into a 100 m-long capillary column (SP™2560, Supelco, Bellefonte, PA, USA) with an internal diameter of 0.25 mm. Helium was used as the carrier gas with a flow rate of 20 cm/s. The temperature of the injector and detector were both set at 260 °C. The oven temperature was initially set at 140 °C for 5 min, and increased from 140 to 240 °C at an increasing rate of 4 °C/min, and finally held at 240 °C for 20 min. 2.6. Determination of lipid productivity The liquid productivity (Plipid) was determined based on the calculation indicated in Eq. (1):
Plipids ðg=l=dÞ ¼
3.1. The effects of media and cultivation conditions on biomass production and composition of C. vulgaris ESP-31 It is known that the growth rate and the biomass composition of microalgae would vary under different medium composition and cultivation conditions (Chen et al., 2011). To determine a suitable medium for the growth and lipid production of C. vulgaris ESP31, three kinds of media of different richness with regard to nitrogen source and nutrient (mainly trace metal ions) content (Table 1) were used to cultivate the microalga in this study, because lipid accumulation in microalgae is often triggered under nitrogen or nutrient limiting conditions (Illman et al., 2000; Li et al., 2008; Solovchenko et al., 2008; Yeesang and Cheirsilp, 2011; Yeh and Chang, 2011). On the other hand, Chlorella sp. can adapt to different growth conditions such as phototrophic, heterotrophic and mixotrophic cultivation (Chen et al., 2011; Cheng et al., 2009; Chiu et al., 2008; Liang et al., 2009; Xiong et al., 2008), and some reports have indicated that the lipid content and productivity could be greatly dependent on those conditions. In general, microalgae could be grown under four different cultivation conditions (Chen et al., 2011; Chojnacka and Marquez-Rocha, 2004; Mata et al., 2010), as described in Table 2. In this work, all four conditions shown in Table 2 were used to grow C. vulgaris ESP-31 to examine which results in the highest biomass and lipid productivity. Fig. 1 shows the of biomass production performance of C. vulgaris ESP-31 grown under different combinations of media (Basal medium, Modified Bristol’s medium, and MBL medium) and cultivation conditions (phototrophic, heterotrophic, mixotrophic and photoheterotrophic cultivation). The inter-correlation of cell growth with medium and cultivation conditions depicted in Fig. 1 shows a biomass concentration of below 0.5 g/l was observed under phototrophic (using 1 g/l NaHCO3 as carbon source) and heterotrophic (using 10 g/l glucose as carbon source) growth. The use of a higher NaHCO3 concentration (1.2–1.6 g/l) for phototrophic cultivation resulted in high pH (10 and 11) in the broth, thereby causing inhibitory effects with regard to the microalgae growth (Yeh et al., 2010). This limited the biomass production to no more than 0.46 g/l when using NaHCO3-amended Basal medium for microalgal growth (Fig. 1). In contrast, when CO2 was used as the carbon source for phototrophic growth on Basal medium, the highest biomass production reached 5.00 g/l (Fig. 1). In phototrophic cultivation, better cell growth was obtained when using a nitrogen-rich medium (i.e., Basal medium and Modified Bristol’s medium) when
Cumulative microalgae biomass production ðgÞ lipid content ð%Þ Working volume ðlÞ cultivation time ðdÞ
ð1Þ
K.-L. Yeh, J.-S. Chang / Bioresource Technology 105 (2012) 120–127
Fig. 1. Biomass production performance of C. vulgaris ESP-31 grown on different media (Basal, Modified Bristol’s and MBL medium) under different cultivation conditions (phototrophic, heterotrophic, mixotrophic and photoheterotrophic cultivation).
compared with growth on nitrogen-poor MBL medium (Fig. 1), indicating that nitrogen source concentration is essential in supporting photoautotrophic growth of the microalgal strain. C. vulgaris ESP-31 cannot grow well under heterotrophic conditions. However, with a light supply, the microalga grew efficiently, as the biomass concentration in photoheterotrophic culture was 15 times higher than that obtained under heterotrophic cultivation on Basal medium and Modified Bristol’s medium (Fig. 1). Mixotrophic growth was also effective, with similar biomass production efficiency to that seen with photoheterotrophic growth (Fig. 1). Apparently, light energy is required for the C. vulgaris ESP-31 strain to efficiently assimilate organic carbon sources. Some C. vulgaris strains seem to display better growth under photoheterotrophic conditions. For instance, Liang et al. (2009) reported that when their C. vulgaris strain was grown on 1% glucose with light (photoheterotrophic condition), the highest biomass production (2 g/l) and lipid productivity (54.0 mg/l/d) achieved were superior to those obtained under heterotrophic conditions with the same glucose concentration. These results are quite similar to what was observed with the C. vulgaris ESP-31 strain. However, there are also some microalgal species that can grow very fast on organic carbon sources in the dark (Cheng et al., 2009; Liang et al., 2009; Xiong et al., 2008), and the glucose metabolism is different under dark and light conditions. Pentose Phosphate Pathway (PPP) is the main pathway under darkness, while under light it is the Embden-Meyerhof Pathway (EMP) (Bashan et al., 2011). Some microalgae species cannot metabolize glucose under anaerobic-dark conditions due to the low levels of the lactate dehydrogenase enzyme; however, there are some species that still cannot assimilate glucose even after having the enzymes (Bashan et al., 2011; Neilson and Lewin, 1974). Photoheterotrophic organisms can only grow when both light and glucose are present, implying that glucose is not the energy source but rather the building material. In photoheterotrophic cultivation, light energy is used as the energy source, which is transformed into chemical energy, such as ATP and NADPH (Chojnacka and Marquez-Rocha, 2004). Our results show that the C. vulgaris ESP-31 strain can use both inorganic and organic carbon sources for growth, but light is necessary in both situations. The results also show that the biomass production is not only
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dependent on growth conditions, but also on the medium used. Therefore, both factors should be taken into account simultaneously to optimize microalgal growth. The main components of microalgae are lipid, protein and carbohydrate. Under common condition, the lipid, protein and carbohydrate content of C. vulgaris is about 14–22%, 51–58%, and 12– 17%, respectively (Spolaore et al., 2006). Fig. 2 shows the biomass composition of C. vulgaris ESP-31 under different growth conditions. Carbohydrate content was about 30–40% in most cultivation conditions with CO2 aeration (phototrophic and mixotrophic condition), while a higher carbohydrate content (about 50–60%) was obtained when using glucose as the only carbon source (heterotrophic and photoheterotrophic condition). Fig. 2 also shows that under all cultivation conditions cell growth on Basal medium (rich in nitrogen and nutrient content) led to lower lipid content (5–22%), while higher lipid content (19–53%) occurred in MBL medium, which has lower nitrogen and nutrient content. The highest lipid content (53%) was obtained when using MBL medium under phototrophic growth. This clearly shows that, as often mentioned in the literature, a deficiency in nitrogen source or nutrients could improve lipid and carbohydrate accumulation (Griffiths and Harrison, 2009; Mata et al., 2010; Yeh and Chang, 2011). The high lipid and carbohydrate content observed in C. vulgaris ESP-31 might due to the cells were collected at stationary phase of the growth. In the exponential growth phase of microalgae, the biomass contains mostly proteins (Mata et al., 2010). However, the nitrogen and nutrient usually deplete at the stationary phase and the lipid/carbohydrate start to accumulate in microalgal cells. Thus, the medium composition and growth conditions are the most critical factor affecting the lipid and carbohydrate content of the microalgae. Furthermore, there is a common trend that higher lipid and carbohydrate content always appears with lower protein content (Fig. 2). This result seems reasonable, since lipid and carbohydrate accumulation usually occurs under nitrogen limiting condition, and so the intracellular proteins may be degraded to supply nitrogen to maintain the metabolic functions in microalgae (Gouveia et al., 2009; Mata et al., 2010). 3.2. The effects of media composition and cultivation conditions on lipid productivity of C. vulgaris ESP-31 According to the biomass composition data (Fig. 2), C. vulgaris ESP-31 has the potential for lipid production, as its lipid content could reach up to 53% under suitable medium and cultivation conditions. Although the results indicate that the use of MBL medium could lead to higher lipid contents, the microalgal growth rate was very low on MBL medium due to its low nitrogen and nutrient content. Therefore, from an engineering perspective, it would be more important and practical to evaluate the lipid production performance by simultaneously considering lipid content and biomass production rate, rather than simply emphasizes the lipid content (Griffiths and Harrison, 2009; Yeh and Chang, 2011). As a result, in this study, lipid productivity (Plipid g/ l/d), which combines the dual effects of lipid content and biomass productivity, was used as the performance index to examine the efficiency of lipid production from the microalgal strain (Chen et al., 2011; Griffiths and Harrison, 2009; Yeh and Chang, 2011). Table 3 compares the lipid productivities obtained under different media and cultivation conditions. Regardless of the media used, the two unfavorable cultivation conditions (i.e., phototrophic culture with NaHCO3 and heterotrophic culture) resulted in very poor lipid productivity (2.7–7.7 mg/l/d) (Table 3). For the rest of the cultivation conditions, the lipid productivity obtained from using Basal medium only varied slightly (56–69 mg/l/d), but for the microalga grown on MBL medium and Modified Bristol’s
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Fig. 2. Biomass composition of C. vulgaris ESP-31 grown under different cultivation conditions (phototrophic, heterotrophic, mixotrophic and photoheterotrophic cultivation) on (a) Basal medium, (b) Modified Bristol’s medium and (c) MBL medium.
medium, the lipid productivity varied significantly (21–105 mg/l/d and 40–144 mg/l/d, respectively) when different cultivation conditions were used (Table 3). Higher lipid productivities were obtained when using glucose as the carbon source in photoheterotrophic or mixotrophic cultures (Table 3). Although the highest lipid content (53%) was obtained under phototrophic growth using CO2 as the carbon source on MBL medium (Figs. 2 and 3), the
highest lipid productivity (143.9 mg/l/d) occurred in the mixotrophic culture on Modified Bristol’s medium (Table 3). This suggests that the best lipid production performance may not necessarily originate from the microalgal biomass with the highest lipid content, whereas the cell growth rate and biomass productivity may play a determining role in the productivity of microalgal lipid. The highest productivity (143.9 mg/l/d) obtained from this study is also among the highest that has yet been reported in the literature (Chen et al., 2011; Mata et al., 2010). Fig. 3 shows the time-course profiles of biomass production, residual nitrogen concentration and lipid content under different media and cultivation conditions. The specific growth rate and nitrogen utilization rate under photoheterotrophic conditions are much higher than under phototrophic ones (on Basal and Modified Bristol’s medium), resulting in higher lipid productivity in photoheterotrophic cultures even though the lipid contents are similar in both conditions (Fig. 3). Moreover, the use of mixotrophic conditions with all medium could obtain the highest nitrogen utilization rate, lipid accumulation rate, lipid content (40–53%) and lipid productivity (67–144 mg/l/d). In all cases, the microalgal lipids were significantly accumulated after the nitrogen deficiency, especially in mixotrophic conditions (Fig. 3). Darzins et al. (2008) examined the fatty acid synthesis pathway in chloroplasts, and mentioned that the conversion of acetyl CoA to malonyl CoA is the key step in fatty acid synthesis. The formation of malonyl CoA from acetyl CoA and CO2 (from HCO 3 ) is catalyzed by acetyl CoA carboxylase (ACCase) (Darzins et al., 2008; Ohlrogge and Browse, 1995). Therefore, when the microalgae assimilate glucose as the carbon source, the culture enters the nitrogen starvation period faster and, with the addition of CO2 under mixotrophic conditions, the rate of lipid accumulation would be stimulated. This may explain why mixotrophic cultures resulted in higher lipid productivities with the microalga. Although using glucose for microalgal growth resulted in greater lipid productivity, the conversion of CO2 to microalgal lipid is more promising from the perspective of CO2 mitigation (Chisti, 2007; Mata et al., 2010). The lipid contents of phototrophic cultures on Basal and Modified Bristol’s media only slightly increased from 18% to 23–27% after nitrogen starvation, while it increased rapidly from 38% to 68% in MBL medium (Fig. 3). This may be because the nitrogen source concentration of MBL medium is much lower than that of the other two media, indicating that the initial nitrogen source concentration might be the key factor influencing the lipid accumulation (Yeh and Chang, 2011). This microalga strain thus still has the potential to produce microalgal lipids for making biodiesel via phototrophic cultivation if an appropriate medium and nitrogen source concentration is used. 3.3. Fatty acid profile of the produced microalgal lipids The performance of microalgal lipid production of C. vulgaris ESP-31 has been described in the earlier sections. It is also equally
Table 3 Lipid productivity of C. vulgaris ESP-31 grown on different media (Basal, Modified Bristol’s and MBL medium) under different cultivation conditions (phototrophic, heterotrophic, photoheterotrophic and mixotrophic cultivation). Medium
Basal Modified Bristol’s MBL
Lipid productivity (mg/l/d) Phototrophic culture (NaHCO3)
Phototrophic culture (CO2)
Heterotrophic culture (glucose)
Photoheterotrophic culture (glucose)
Mixotrophic culture (glucose and CO2)
2.7 ± 0.5 3.7 ± 0.4
56.2 ± 2.9 40.2 ± 0.8
3.5 ± 0.5 3.5 ± 0.1
69.0 ± 2.3 115.4 ± 24.7
67.4 ± 2.5 143.9 ± 0.8
6.3 ± 0.2
51.2 ± 5.5
7.7 ± 0.4
20.7 ± 3.9
104.9 ± 7.2
Data shown are averages of two runs ± SD (standard deviation).
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6
Biomass production (g/L)
Phototrophic (CO2)
Basal medium
Modified Bristol's medium
MBL medium
Photoheterotrophic Mixotrophic
5
4
3
2
1
Nitrate concentration (mg/L)
0 800
600
400
200
Lipid content (wt. %/biomass)
0 70 60 50 40 30 20 10 0 0
4
8
12
16
20
0
4
Time (d)
8
12
Time (d)
16
20
0
4
8
12
16
20
Time (d)
Fig. 3. Time-course profile of biomass production, nitrogen concentration and lipid content of C. vulgaris ESP-31 grown on Basal medium, Modified Bristol’s medium and MBL medium under different cultivation conditions. Data shown are the average of two runs ± SD (standard deviation).
important to determine the quality of the microalgal lipids thus produced to assess the feasibility of using them for biodiesel synthesis. The raw materials used for making biodiesel need to meet some standard regulations issued by local governments. For instance, the ASTM Biodiesel Standard D6751 is executed in the United States, while in European Union vehicles using biodiesel need to follow regulations based on the Standard EN 14214 (Chisti, 2007). The European biodiesel standards have a restriction limiting the FAMEs content with four and more double bonds to 1 mol%, and the linolenic acid methyl ester (C18:3) content to 12 mol% (Chisti, 2007). In addition, lipids with fatty acids containing 16–18 carbon atoms are considered suitable for producing biodiesel (Huang et al., 2010). Therefore, analyzing the fatty acid profile of microalgal lipids produced from C. vulgaris ESP-31 is necessary to assess whether the quality is suitable for biodiesel synthesis (Chisti, 2007; Huang et al., 2010). Table 4 shows the profiles of lipid content and fatty acid composition of C. vulgaris ESP-31 under different medium and cultivation conditions. The main fatty acids in C. vulgaris ESP-31 were palmitic
acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3). Among those, palmitic acid and oleic acid were the two most predominant fatty acid components, accounting for 50–62% of total fatty acids present in the microalgal lipids (Table 4). This lipid profile is in accord with that reported in the literature, indicating that the main fatty acids present in the lipid of Chlorella sp. are normally short chain fatty acids (C14–C18) (Huang et al., 2010). There is a clear trend that for microalgal cells with over 40% lipid content, higher C16:0 and C18:1 content was observed in the microalgal lipids, while for the microalgal lipids with the content under 30%, more C16:0 and C18:2 fatty acids appeared (Table 4). This trend can also be clearly observed in Fig. 4. The relative content of C16:0 increased from 18% to 31% and an increase from 3% to 33% was observed in fatty acid C18:1 when lipid content of microalgae increased from 13% to 63%, whereas the relative content of C18:2 reduced from 26% to 16% for microalgal lipid with the same lipid content range. Similar results were reported by other researchers (Darzins et al., 2008), as they found that in green algae the proportion of
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Table 4 The fatty acids composition of C. vulgaris ESP-31 grown in different media (Basal, Modified Bristol’s and MBL medium) and different cultivation conditions (phototrophic, mixotrophic and photoheterotrophic cultivation). Medium
Cultivation condition
Basal
Phototrophic (CO2)
56.2 ± 2.9
Basal
Photoheterotrophic
69.0 ± 2.3
Basal
Mixotrophic
67.4 ± 2.5
Modified Bristol
Phototrophic (CO2)
40.2 ± 0.8
Modified Bristol
Photoheterotrophic
115.4 ± 24.7
Modified Bristol
Mixotrophic
143.9 ± 0.8
MBL
Phototrophic (CO2)
51.2 ± 5.5
MBL
Photoheterotrophic
20.7 ± 3.9
MBL
Mixotrophic
Fatty acid content (wt.%/dry weight of biomass)a or relative content (%)b
Lipid productivity (mg/l/d)
104.9 ± 7.2
Total
C16:0
C16:1
C18:0
C18:1
C18:2
C18:3
Others
23.89a 100b 13.14a 100b 45.44a 100b
5.24 21.93 2.16 16.44 13.09 28.81
0.06 0.25 0.28 2.13 0.57 1.25
2.39 10.00 1.96 14.92 3.03 6.67
3.31 13.86 0.48 3.65 11.29 24.85
6.16 25.78 3.31 25.19 9.36 20.60
1.18 4.94 0.97 7.38 3.08 6.78
5.55 23.23 3.98 30.29 5.02 11.05
21.72a 100b 30.18a 100b 57.61a 100b
3.89 17.91 6.87 22.76 16.59 28.80
0.04 0.18 0.23 0.76 0.38 0.66
2.38 10.96 2.38 7.89 3.58 6.21
1.23 5.66 8.06 26.71 18.94 32.88
5.03 23.16 7.06 23.39 9.29 16.13
1.98 9.12 1.03 3.41 2.79 4.84
7.17 33.01 4.55 15.08 6.04 10.48
62.95a 100b 40.62a 100b 49.92a 100b
19.71 31.31 10.75 26.46 14.63 29.31
0.06 0.10 0.27 0.66 0.50 1.00
4.44 7.05 2.87 7.07 3.83 7.67
18.48 29.36 10.43 25.68 14.63 29.31
10.04 15.95 7.26 17.87 8.17 16.37
3.67 5.83 3.60 8.86 3.15 6.31
6.55 10.41 5.44 13.39 5.01 10.04
produced by C. vulgaris ESP-31 contained over 60–68% of saturated and monounsaturated fatty acids, thereby being suitable for applications in the biodiesel industry.
Relative content of fatty acid (%)
35 30
Acknowledgements
25
The authors gratefully acknowledge financial support from Taiwan’s National Science Council under grant numbers NSC 1003113-E-006-016- and NSC 99-3113-P-110-001-. The support from the Top University grant of National Cheng Kung University is also appreciated.
20 15 10
C16:0 C18:1 C18:2
5 0
0
10
20
30
40
50
60
References
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Lipid content (wt. % per biomass) Fig. 4. The relative content of fatty acids (C16:0, C18:1 and C18:2) at different lipid content of C. vulgaris ESP-31 grown in different media (Basal, Modified Bristol’s and MBL medium) and different cultivation conditions (phototrophic, mixotrophic and photoheterotrophic cultivation).
polyunsaturated C18:2 fatty acid in the microalgal biomass tends to decrease after nitrogen starvation. In this study, the lipids produced consisted of over 60–68% of saturated and monounsaturated fatty acids (palmitic acid (C16:0), stearic acid (C18:0) and oleic acid (C18:1)), which are considered suitable for synthesizing biodiesel (Sheehan et al., 1998). Hence, the C. vulgaris ESP-31 strain seems to be an excellent candidate as biodiesel feedstock. 4. Conclusions This study demonstrates the feasibility of using an indigenous microalga C. vulgaris ESP-31 as feedstock for biodiesel production. The cell growth and lipid accumulation are greatly affected by medium composition and cultivation conditions. Higher biomass production (2–5 g/l) was obtained using nitrogen-rich medium under various growth conditions, whereas higher lipid content (20–53%) was achieved by using a nitrogen-limiting MBL medium. The highest lipid productivity (144 mg/l/d) was obtained from mixotrophic cultivation on Modified Bristol’s medium. The lipid
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