Bioresource Technology 173 (2014) 406–414

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Growth and lipid accumulation characteristics of Scenedesmus obliquus in semi-continuous cultivation outdoors for biodiesel feedstock production Pingzhong Feng a,1, Kang Yang a,1, Zhongbin Xu a, Zhongming Wang a, Lu Fan b, Lei Qin a, Shunni Zhu a, Changhua Shang a, Peng Chai a, Zhenhong Yuan a,⇑, Lei Hu c a

Key Laboratory of Renewable Energy, Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, China Microalgae Laboratory, School of Resource & Environmental Engineering, Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Hubei University of Technology, Wuhan 430068, China c Jiangsu Key Laboratory for Biomass-based Energy and Enzyme Technology, Huaiyin Normal University, Huaian 223300, China b

h i g h l i g h t s  Scenedesmus obliquus is a promising organism for biofuel feedstock production. 1

 S. obliquus can grow fast (2.09 day

) and store lipids up to 49.6% of dry weight.

 Outdoor saturated fatty acids content in cells was higher than that achieved indoors.  The cells reached high lipid contents (>46%) in semicontinuous cultivation outdoors. 1

 High lipid productivities (151–193 mg L

a r t i c l e

day1) were also obtained outdoors.

i n f o

Article history: Received 11 August 2014 Received in revised form 22 September 2014 Accepted 23 September 2014 Available online 30 September 2014 Keyword: Microalgal culture Semicontinuous cultivation Lipid accumulation Biodiesel Scenedesmus obliquus

a b s t r a c t In an effort to identify suitable microalgal species for biodiesel production, seven species were isolated from various habitats and their growth characteristics were compared. The results demonstrated that a green alga Scenedesmus obliquus could grow more rapidly and synthesize more lipids than other six microalgal strains. S. obliquus grew well both indoors and outdoors, and reached higher lmax indoors than that outdoors. However, the cells achieved higher dry weight (4.36 g L1), lipid content (49.6%) and productivity (183 mg L1 day1) outdoors than in indoor cultures. During the 61 days semi-continuous cultivation outdoors, high biomass productivities (450–550 mg L1 day1) and lmax (1.05–1.44 day1) were obtained. The cells could also achieve high lipid productivities (151–193 mg L1 day1). These results indicated that S. obliquus was promising for lipids production in semi-continuous cultivation outdoors. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction The crude oil might be depleted within 40 years, alternative fuels have to be developed to drive our transportation systems, and biodiesel appears to be the most promising biofuel of the future (Farrell et al., 2006; Rodolfi et al., 2009). Biodiesel is renewable, non-toxic, and biodegradable, it can be used in existing diesel engines without modifying the engines, and can be blended in at ⇑ Corresponding author. Tel.: +86 20 87057705. E-mail address: [email protected] (Z. Yuan). These authors contributed to the work equally and should be regarded as co-first authors. 1

http://dx.doi.org/10.1016/j.biortech.2014.09.123 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

any ratio with petroleum diesel (Vicente et al., 2004). However, the development of biodiesel industry is severely limited by the supply of feedstock, namely soybean oil, canola oil (Ma and Hanna, 1999). Due to the limitation of available agriculture land and irrigation water supply, the production of these oil crops cannot sustain the biodiesel production, other sources of plant oil have to be developed as feedstock for biodiesel. In the past ten years, microalgae have received increasing attentions for biodiesel feedstock production (Chisti, 2007; Hu et al., 2008). The advantages of microalgae over other oil plants as a source of biodiesel are numerous, such as: (1) higher growth rates, lipid contents, photosynthetic efficiency and carbon dioxide fixation rates (Chisti, 2007; Hu et al., 2008); (2) microalgae-based

P. Feng et al. / Bioresource Technology 173 (2014) 406–414

biodiesel is renewable, non-toxic and contributes no net increase of carbon dioxide or sulfur to the atmosphere (Vicente et al., 2004); (3) much less land is needed to culture microalgae compare to conventional oil crops (Sheehan et al., 1998; Chisti, 2007); (4) the residual algal biomass after oil extraction may be used as feed or fertilizer, or fermented to produce ethanol or methane (Rodolfi et al., 2009). Although microalgae represents a promising alternative for biodiesel production, and has already been proven to be successful in laboratory scale operations, no commercial production of biodiesel from microalgae is available (Sheehan et al., 1998; Chisti, 2007; Hu et al., 2008). This is mainly caused by the high overall costs of algal biofuel production (Sheehan et al., 1998; Chisti, 2007). Previous reports indicated that microalgal species (Sheehan et al., 1998; Chisti, 2007), algal cultivation (Chisti, 2007), algal biomass harvesting (Sheehan et al., 1998), oil extraction (Sheehan et al., 1998; Sostaric et al., 2012) and industrial-scale transesterification (Klofutar et al., 2010; Likozar and Levec, 2014) are some of the key aspects affecting the economic feasibility of microalgae for biodiesel production. To enhance the economic feasibility of producing microalgae-based biodiesel, the first step is to select appropriate microalgal species for producing biodiesel from microalgae (Griffiths and Harrison, 2009). Promising microalgae species are characterized by high growth rates and lipid contents, as well as suitability for massive cultivation, even for growing outdoors under natural sunlight (Chisti, 2007; Nascimento et al., 2013; El-Sheekh et al., 2013; Feng et al., 2011). Many promising strains proposed for biodiesel production were Chlorella sp. (Lv et al., 2010; Liu et al., 2010; Feng et al., 2012), Nannochloropsis (Rodolfi et al., 2009; Moazami et al., 2012), Chlorococcum (Feng et al., 2014) and Scenedesmus spp. (Ho et al., 2010). However, among those microalgal strains reported by previous studies, only a few strains had been demonstrated the feasibility of meeting the requirements of large scale cultivation by using natural sunlight, such as Chlorella, Nannochloropsis, etc. Li et al. (2007) reported that the lipid productivity reached 0.7 g L1 day1, when Chlorella protothecoides was cultured in an 8000 L stirred tank bioreactor under heterotrophic condition. Chlorella zofingiensis was considered as an appropriate specie for massive cultivation to produce biodiesel feedstock, because its lipid content could reach a high level of 54.5% outdoors (Feng et al., 2011). The results from the study of Moazami et al. (2012) revealed that the maximum lipid content and hydrocarbon productivity of Nannochloropsis cultured in a 2000 L raceway pond could reach 52% and 70 mg L1 day1, respectively. Improving the biomass productivity and lipid productivity is the important avenue to increase the economic feasibility of microalgae-based biodiesel production. Stress conditions usually enhance the microalgal cell lipid contents, but limit the cell growth, leading to low biomass productivity and low overall lipid productivity (Dragone et al., 2011). Therefore, two-phase cultivation process was considered as a suitable strategy that could achieve the best combination of high lipid content and biomass production: a nutrient-sufficient phase to cultivate high biomass concentration followed by a nutrient limitation phase to induce lipid accumulation (Rodolfi et al., 2009). In this study, a green microalgal strain Scenedesmus obliquus was selected, and the growth and lipid accumulation potential were investigated. To optimize the growth conditions of S. obliquus, the dry weight and growth rate were monitored with respect to time when the microalga was grown under different mediums and cultivation conditions. The growth rate, lipid content/productivity and fatty acid profile of S. obliquus cultured outdoors and indoors were also studied and compared. In addition, to evaluate

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the feasibility of cultivating outdoors, S. obliquus was cultured for 61 days by two-phase process in semi-continuous cultivation outdoors. The present study aimed to identify the optimal conditions for the cell growth and lipid accumulation of S. obliquus, and to evaluate the potential of using this alga to produce biodiesel feedstock. 2. Methods 2.1. Algal strain The microalgal species (S. obliquus, Scenedesmus dimorphus, Scenedesmus sp., Chlorella vulgaris, Chlorella sp. 1, Chlorella sp. 2 and Chlorococcum sp.) used in this study were collected from freshwater habitats in southern China and preserved in Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences. BG11 culture medium was used for maintenance and seed cultivation of the organisms. 2.2. Algal cultivation conditions in experiments 2.2.1. The growth and lipid accumulation characteristics of the seven microalgal strains To investigate the growth and lipid accumulation characteristics of those seven microalgal strains, cells at exponential phase were respectively inoculated into BG-11 medium (in growth experiments) and BG-110 medium (nitrogen-free) (in lipid accumulation experiments) in columns (clear glass; diameter: 4.5 cm; culture volume: 500 ml), and cultured under continuous fluorescent light (200 lmol m2 s2) at 25 °C. Constant aeration ðV air =V CO2 ¼ 98 : 2Þ at about 0.6 L min1 was supplied by utilizing air pump. The dry weight and lipid content were measured in the two experiments, and the specific growth rate (lmax), lipid productivity and biomass productivity were also calculated. 2.2.2. Effects of different conditions on the growth of S. obliquus S. obliquus cells grown at exponential phase were inoculated into 0.5 L glass columns (clear glass; diameter: 4.5 cm; culture volume: 500 ml) indoors by investigating the effects of culture conditions on the growth of S. obliquus. Constant aeration (air with CO2) at about 0.6 L min1 was supplied by utilizing air pump. Continuous fluorescent light (fluorescent tubes) was also provided. The concentration of CO2 (0–20%) and light intensity (20– 400 lmol m2 s2) were set according to the experimental requirements. The temperature for cultivation was 25 °C. The dry weight was measured in the experiments, and the specific growth rate (lmax) and biomass productivity were also calculated. The experiments were carried out according to the orders below: (1) Effects of different culture mediums on growth: the BG11, modified BG-11 (1 g L1 of NaNO3, 0.06 g L1 of K2PO43H2O), f/2 and BBM. Constant aeration ðV air =V CO2 ¼ 98 : 2Þ; and continuous fluorescent light (200 lmol m2 s2) were supplied. (2) Effects of different light intensities on the growth: 20, 100, 200 and 400 lmol m2 s2. Constant aeration ðV air =V CO2 ¼ 98 : 2Þ was supplied by utilizing air pump. The cultural medium was modified BG-11 based on the experimental results above. (3) Effects of different CO2 concentrations on the growth: 0 (only air), 0.5%, 5%, 10% and 20% CO2 ðV CO2 =V air þCO2 Þ. According to the experimental results above, the cultural medium was modified BG-11, and constant aeration ðV CO2 =V air ¼ 5 : 95Þ and continuous fluorescent light (400 lmol m2 s2) were supplied.

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2.2.3. The comparisons between the indoor and outdoor cultivations of S. obliquus in 80 L flat plate photobioreactors To further evaluate the potential of S. obliquus for biodiesel feedstock production, the growth and lipid accumulation of the strain grew in 80 L FPPs (flat plate photobioreactors) indoors and outdoors were investigated and compared. The photobioreactors used in the experiments was FPP. The modules used in the experiments were 1.5 m long, 1.2 m high and 5 cm thick, with a culture volume of 80 L. For mixing, compressed air with 5% CO2 at about 20 L min1 was bubbled at the bottom of the reactor through a perforated plastic tube. The FPPs were made of glass and stainless steel. The temperature for indoor cultivation was 25 °C. Continuous fluorescent light (fluorescent tubes) with a light intensity of 400 lmol m2 s2 was also supplied for the indoor cultivation in FPPs, while the illumination provided for outdoor cultivation was only the natural sunlight. The cultural medium used in growth experiments was modified BG-11 with nitrogen sufficient, and modified BG-11 with nitrogen free was used for lipid accumulation in the experiments. 2.2.4. Culturing S. obliquus by two-phase process in semi-continuous cultivation outdoors An important aspect of commercially successful algal culture is the ability to grow the algae in continuous or semi-continuous culture for long periods (Moheimani and Borowitzka, 2006). That can optimize the use of capital intensive culture systems and also reduce labor costs (Sheen et al., 1998). To evaluate the lipid production potential of S. obliquus under natural sunlight, the cells were grown outdoors in 80 L FPPs under nitrogen sufficient and free conditions, respectively. The experiments were carried out between Sep. 15 and Nov. 14. Constant aeration with a aeration rate about 20 L min1 ðV air =V CO2 ¼ 95 : 5Þ was provided by utilizing air pump. The illumination in the experiments was only the natural sunlight. The cultural medium was modified BG-11 (Nitrogen sufficient and nitrogen free medium was used for growth phases and lipid accumulation phases in the experiments, respectively).

where DWi and DW0 is the dry weight on day ti and day t0 (the first day), respectively. Where Ci and C0 is the lipid content on day ti and t0 (the first day), respectively. (3) The following equation was used for calculating the biomass productivity (Pbiomass):

Pbiomass ¼ ðDWi  DW0 Þ=ðt i  t 0 Þ where DWi and DW0 is the dry weight on day ti and t0 (the first day), respectively. 2.3. Dry weight determination Algal cells were collected by filtering through a pre-weighed Whatman GF/C filter paper and washed with equal amount of deionized water, then dried in an oven at 105 °C overnight. 2.4. Total lipids measurement and fatty acid profiles analysis Total lipids were extracted from algal cells following the method of Bligh and Dye (1959). The lipid content was measured gravimetrically and expressed as percentage of the dry weight. Fatty acid methyl esters (FAMEs) compositions were determined by the direct transesterification of algae biomass and analyzed by GC methodology. FAMEs were prepared by acid transesterification according to the method of Indarti et al. (2005). After that, the FAMEs were analyzed by using a gas chromatograph (GC-2010, Shimadzu, Japan) with a flame ionization detector. Nitrogen was used as carrier gas with a flow rate of 1.2 ml min1. The injector and detector temperature were set at 250 °C and 280 °C, respectively. The initial temperature of column was set at 140 °C, and increased to 180 °C at a rate of 10 °C min1 followed by a rise to 230 °C with 1 °C min1, and then the temperature was kept constant at 230 °C for 10 min. Ultimately, the fatty acids were recorded as percentages of total fatty acids. 3. Results and discussion

2.2.5. Determination growth and lipid of microalgae 3.1. Biomass and lipid concentration and productivity (1) The specific growth rate (lmax) in exponential phase of algal growth was calculated by using the equation below: 1

lmax ðday Þ ¼ ðln x2  ln x1 Þ=ðt2  t1 Þ where t1 and t2 are defined as dry weight (g L1) at time t1 and t2 in exponential growth phase, respectively. (2) The lipid productivity (Plipid) was calculated according to the following formula: 1

Plipid ðmg L1 day Þ ¼ ðDWi  C i  DW0  C0 Þ=ðt i  t 0 Þ

As shown in Table 1, among the seven microalgal strains, S. obliquus achieved the highest biomass productivity (586 mg L1 day1), lipid productivity (207 mg L1 day1), specific growth rate (1.68 day1) and biomass concentration (4.2 g L1). In addition, the lipid content of S. obliquus also reached a high level (48.2% of dry cell weight), even though it was slightly less than that of Chlorococcum sp. (52.1% of dry cell weight). Relatively high costs of microalgae-based biodiesel production due to low lipid productivity have been one of the major obstacles impeding commercial microalgal oil production (Sheehan et al., 1998). In order to

Table 1 Comparison of lmax, biomass concentration, biomass productivity, lipid content and lipid productivity among seven microalgae strains.a

a b c

Strain

Specific growth rate lmax (day1)

Biomass concentration (g L1)b

Biomass productivity (mg L1 day1)b

Lipid content (%)c

Lipid productivity (mg L1 day1)c

Scenedesmus obliquus Scenedesmus dimorphus Scenedesmus sp. Chlorella vulgaris Chlorella sp. 1 Chlorella sp. 2 Chlorococcum sp.

1.68 ± 0.02 1.43 ± 0.03 1.30 ± 0.02 1.48 ± 0.04 1.51 ± 0.03 1.63 ± 0.01 1.47 ± 0.02

4.20 ± 0.12 2.8 ± 0.09 2.2 ± 0.10 2.18 ± 0.18 2.65 ± 0.13 1.36 ± 0.08 3.1 ± 0.13

586 ± 21 386 ± 18 300 ± 27 297 ± 20 364 ± 25 180 ± 13 429 ± 28

48.2 ± 2.1 35.6 ± 1.9 45.7 ± 4.2 36.0 ± 1.5 37.1 ± 2.3 42.3 ± 2.6 52.1 ± 4.7

207 ± 10 115 ± 11 159 ± 13 109 ± 9 127 ± 10 142 ± 9 180 ± 12

Data are reported as mean ± standard deviation of triplicates. On day 8 (the last day of the cultivation; algal cells were cultured for 7 days in nitrogen sufficient medium). On day 9 (the last day of the cultivation; algal cells were cultured for 8 days in nitrogen free medium).

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increase the economic feasibility of microalgal biodiesel production, the screening of microalgal species with high lipid productivity is of great importance (Lv et al., 2010). Obviously, S. obliquus was more promising compare to other six candidates for producing biofuel feedstock. Therefore, S. obliquus was selected for further characterization in the following experiments. 3.2. Effects of different conditions on the growth of S. obliquus In order to enhance the specific growth rate and biomass productivity, the growth conditions of S. obliquus were preliminarily optimized. Culture medium was optimized firstly, followed by light intensity and then CO2 concentration (Table 2). As it is shown in Table 2, S. obliquus grew better in modified BG11 than in the other three mediums. The possible reasons were that: (1) Nitrate and phosphate are almost the most important nutrients for microalgal growth in autotrophic cultivation. In the experiments, modified BG-11 contained more appropriate nitrate (1.0 g/L) and phosphate (0.06 g/L) concentrations than those in BG-11 (1.5 g/L of nitrate, 0.04 g/L of phosphate), f/2 (0.075 g/L of nitrate, 0.00565 g/L of phosphate) and BBM (0.25 g/L of nitrate, 0.25 g/L of phosphate) mediums for the growth of S. obliquus; (2) the vitamins (B1, B12 and Biotin) contained in f/2 and BBM mediums might also affect the algal growth. The specific growth rate and biomass productivity of S. obliquus showed increasing trends with the increase of light intensity in the experiments (Table 2). In this section, we intended to find out the growth characteristics and the growth potential of S. obliquus under actual indoor conditions. Normally, 400 lmol m2 s2 is a very high light intensity level for regular fluorescent tubes. It is difficult to obtain very high light intensity by regular fluorescent tubes. Moreover, the cost of large scale indoor cultivation may be also an important limiting factor. Therefore, it was trivial to increase the light intensity even further in the experiment. In addition, among the five CO2 concentrations applied, the highest specific growth rate and biomass productivity of S. obliquus were achieved in the presence of 5% CO2. A plausible explanation for the presence of an optimum CO2 concentration (5%) in the experiments is that as CO2 increases, unutilized CO2 will be converted to H2CO3 thereby reducing the pH of the medium and, in

turn, affecting the algal growth. By contrast, when the CO2 level is low algal growth will be inhibited by the low carbon source (Kaewkannetra et al., 2012). Therefore, the optimal culture medium, light intensity and CO2 concentration were concluded as modified BG11, 400 lmol m2 s2 and 5% CO2 respectively, since S. obliquus cells under those conditions grew obviously faster and achieved higher biomass productivity and biomass concentration than under other conditions in each experiment. As a result, the maximum biomass productivity of 763 mg L1 day1 was observed under optimum conditions, with the highest biomass concentration (dry weight) of 5.44 g L1 on the 8th day of the culture. A high maximum specific growth rate of 2.09 day1 was also obtained under optimum conditions. High lipid productivity is a key desirable characteristic of a species for biodiesel production (Griffiths and Harrison, 2009). The lipid productivity is related to both biomass productivity and lipid content (Lv et al., 2010). Oleaginous microalgae with high cell growth and biomass productivity can increase the economic feasibility for producing biodiesel. In this study, the maximum biomass productivity (763 mg L1 day1) and the highest specific growth rate (2.09 day1) achieved are higher than that obtained from many other oleaginous microalgae strains, such as Pleurochrysis carterae (231 mg L1 day1 and 0.599 day1) (Moheimani and Borowitzka, 2007), S. obliquus CNW-N (280 mg L1 day1) (Ho et al., 2010), Scenedesmus obtusiusculus (520 mg L1 day1 and 0.38 day1) (Toledo-Cervantes et al., 2013), Chlorella pyrenoidosa SJTU-2 (144 mg L1 day1 and 0.993 day1) (Tang et al., 2011), etc. Therefore, the strain selected in this study has promising potential for microalgae-based biodiesel production in the future. 3.3. The comparisons between the indoor and outdoor cultivations of S. obliquus in 80 L flat plate photobioreactors 3.3.1. Specific growth rate, biomass concentration and biomass productivity S. obliquus cells grown indoors at exponential phase were inoculated into 80 L FPPs with an initial dry weight of 0.164 g L1 indoors and outdoors, respectively. The culture medium was Modified BG11 (nitrogen sufficient). Fig. 1 shows the changes of dry

Table 2 Optimization of culture conditions for Scenedesmus obliquus.a Conditions

Specific growth rate lmax (day1)

Final biomass concentration (g L1)b

Biomass productivity (mg L1 day1)b

1.71 ± 0.05 1.9 ± 0.08 0.90 ± 0.06 1.08 ± 0.04

4.51 ± 0.12 4.66 ± 0.15 1.84 ± 0.08 2.15 ± 0.07

630 ± 21 651 ± 29 248 ± 19 293 ± 21

Light intensity (lmol m2 s2) 20 0.82 ± 0.03 100 1.53 ± 0.06 200 1.9 ± 0.08 400 1.97 ± 0.09

1.68 ± 0.10 3.25 ± 0.14 4.66 ± 0.15 4.98 ± 0.17

226 ± 19 450 ± 22 651 ± 29 697 ± 30

CO2 tolerance Air 0.5% 5% 10% 20%

2.36 ± 0.13 4.83 ± 0.18 5.44 ± 0.17 4.92 ± 0.15 1.85 ± 0.11

323 ± 20 675 ± 31 763 ± 33 686 ± 29 250 ± 24

Medium BG11 Modified BG11c f/2 BBM

a

1.37 ± 0.02 1.89 ± 0.08 2.09 ± 0.09 1.91 ± 0.07 1.04 ± 0.05

Data are expressed as mean ± standard deviation of triplicates. On day 8 (the last day of the cultivation; algal cells were cultured for 7 days.); initial dry weight was 0.1 g/L. c The modified BG11 medium contains 1 g L1 of NaNO3 and 0.06 g L1 of K2PO43H2O. The other ingredients in the modified BG11 are the same as those in BG11 medium. b

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Fig. 1. Dry weight and biomass productivity of Scenedesmus obliquus cultured in 80 L flat plate photobioreactors indoors and outdoors. Data are reported as mean ± standard deviation of triplicates.

Fig. 2. Lipid content and productivity of Scenedesmus obliquus cultured in 80 L flat plate photobioreactors indoors and outdoors. Data are reported as mean ± standard deviation of triplicates.

weight (biomass concentration) and biomass productivity during culture period of S. obliquus under indoor and outdoor conditions. The results show that S. obliquus grew well in 80 L FPPs under both indoor and outdoor conditions. As shown in Fig. 1a, S. obliquus cells cultured in 80 L FPPs adapted both cultural indoor and outdoor conditions quickly with short lag periods. Compared with the outdoor cultivation, the alga grew more rapidly in the indoor cultures during the first six days. Moreover, S. obliquus cells grown indoor reached a higher specific growth rate of 1.67 day1 than 1.35 day1 obtained outdoors (Table 3). However, the cells in outdoor cultures somewhat surpassed the indoor cultures on day 7 and achieved a higher final dry cell weight of 4.36 g L1 at the end of the experiments than 4.11 g L1 of indoor cultures (Table 3). In addition, since no obvious sign of the cessation of growth on day 9 was observed, it was predicted that much higher final dry cell weight could be obtained, if the cultures were maintained for additional days. It was clearly observed that the biomass productivity tended to increase in the first few days, then gradually leveled off (Fig. 1b). The maximum biomass productivity (838 mg L1 day1) of S. obliquus occurred on day 3 during indoor cultivation, while under outdoor conditions the maximum biomass productivity was observed on day 7 (598 mg L1 day1) (Fig. 1b). However, in the end of the experiment (on day 9 of the culture), the biomass productivity reached 525 mg L1 day1 outdoors and 493 mg L1 day1 indoors, respectively (Table 3). Such high biomass productivity was also higher compared to those of some other oleaginous microalgae

reported previously (Moheimani and Borowitzka, 2007; Ho et al., 2010; Toledo-Cervantes et al., 2013; Tang et al., 2011), indicating that S. obliquus could adapt outdoor conditions quickly and has promising potential for outdoor cultivation. The continuously illumination (no circadian rhythms) might be an important reason why the alga grew more rapidly indoors in the first few days. In addition, the initial cell concentration was very low (initial dry weight of 0.164 g L1). The outdoor light intensity can reach 2000 lmol m2 s2. It was likely that cells at such low concentration received too much light due to the lack of self-shading, which might lead to photoinhibition. Furthermore, some other factors, such as the fluctuating temperature, contamination, etc., might influence the algal growth outdoors. As a result, the cells cultured indoors grew more rapidly and reached higher biomass productivity in the first few days. However, as the cell concentration increasing (the dry weights were both above 3 g L1 on day 7 indoors and outdoors), it might cause significant self shading among the cells, leading single cell indoors to be exposed under very low light intensity condition. Though continuously illumination was supplied in indoor cultivation, the low light intensity still limited the growth. Therefore, higher dry weight and biomass productivity were achieved outdoors. Fig. 1b also showed that there was almost no biomass productivity decrease in 6 days outdoors. That may primary due to the cells grew slowly in the beginning, leading to low dry weight, then it grew very fast (high dry weight was achieved) in outdoor

Table 3 Specific growth rate, dry weight, biomass productivity, lipid content and productivity of Scenedesmus obliquus cultured in 80 L FPPs indoors and outdoors.a

Indoor Outdoor a b c

Specific growth rate lmax (day1)

Dry weight (g L1)b

Biomass productivity (mg L1 day1)c

Lipid content (%)c

Lipid productivity (mg L1 day1)c

1.67 ± 0.04 1.35 ± 0.02

4.11 ± 0.29 4.36 ± 0.18

493 ± 33 525 ± 57

40.1 ± 0.9 49.6 ± 0.8

115 ± 34 183 ± 25

Data are reported as mean ± standard deviation of triplicates. Final biomass concentration. On day 9 (the last day of the cultivation; algal cells were cultured for 8 days).

P. Feng et al. / Bioresource Technology 173 (2014) 406–414 Table 4 Fatty acid profile of Scenedesmus obliquus cultured in 80 L FPPs indoors and outdoors.a Fatty acids

C14:0 C15:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3b C20:0 C20:1 Others SFAsc MUFAsd PUFAse C16/C18 fatty acids

Contents (% of total fatty acids) Indoors

Outdoors

0.92 ± 0.02 0.69 ± 0.00 21.69 ± 0.61 4.05 ± 0.20 7.11 ± 0.32 23.84 ± 0.96 22.94 ± 0.90 13.02 ± 0.46 0.83 ± 0.00 1.95 ± 0.00 2.96 ± 0.52 31.24 ± 1.03 29.84 ± 0.88 35.96 ± 0.76 92.65 ± 1.44

0.76 ± 0.01 0.83 ± 0.00 26.53 ± 0.51 8.31 ± 0.11 10.72 ± 0.42 29.48 ± 1.11 16.41 ± 0.73 4.09 ± 0.37 1.79 ± 0.00 0.92 ± 0.00 0.16 ± 0.41 40.63 ± 0.92 38.71 ± 1.01 20.5 ± 1.11 95.54 ± 1.32

a

Data are expressed as mean ± standard deviation of triplicates. 18:3 can be either 18:3 (n  3) or 18:3 (n  6), currently, it is not clear which fatty acid is presented in this species. c Saturated fatty acids fatty acids. d Monounsaturated fatty acids. e Polyunsaturated fatty acids. b

cultivation. Therefore, the outdoor biomass productivity calculated according to the equation increased obviously from day 1 to day 5, and almost no decrease was observed before day 8 of the culture. 3.3.2. Lipid content and productivity The changes of lipid contents and productivities of S. obliquus under these conditions were shown in Fig. 2. The cellular lipid contents increased obviously after inoculating into nitrogen free medium in the first few days, under both indoor or outdoor conditions (Fig. 2a). The lipid content of S. obliquus increased a little faster outdoors than that under indoor condition. The cells grew outdoors achieved higher lipid content (49.6%) on day 9 compared to that (40.1%) obtained indoors. As shown in Table 3, S. obliquus cells reached a high lipid productivity of 115 mg L1 day1 indoors on day 9, but a higher lipid productivity of 183 mg L1 day1 was achieved outdoors. Fig. 2b shows that the lipid productivities in outdoor culture were always higher than those obtained from indoor culture during the

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experiments. This result correlates to the fact that the daily average light intensity was (about 1500–2000 lmol m2 s2) markedly higher than that (400 lmol m2 s2) of indoor condition. In addition, the initial cell concentration of 4.05 g L1 (dry cell weight) was a very dense cell culture and caused significant self shading among the cells. As a result, high light intensity in outdoor conditions obviously enhanced the efficiency of lipid accumulation in S. obliquus cells. This result is in agreement with previous reports by Hu et al. (2008) and Rodolfi et al. (2009), who suggested that lipid storage (mainly triacylglycerols) can be increased by increasing light intensity. 3.3.3. Fatty acid profile The most important properties of biodiesel include ignition quality, viscosity, cold flow and oxidative stability, which are largely determined by the structure of component fatty acid esters (Knothe 2005; Hu et al., 2008). Therefore, to increase the feasibility of microalgae-based biodiesel production, it is also important to obtain microalgal species with appropriate lipid compositions. The lipid profile of S. obliquus cultured indoors and outdoors in 80 L FPPs for 8 days was shown in Table 4. As illustrated in Table 4, C16:0, C16:1, C18:0, C18:1, C18:2 and C18:3 were the major fatty acids of S. obliquus, which accounted for more than 90% of total fatty acids. The portion of C16 and C18 fatty acids group from S. obliquus cultivated outdoors (95.54% of total fatty acids) was a little higher than that (92.65% of total fatty acids) obtained indoors. In general, C16 and C18 fatty acids were considered as the ideal components for biodiesel production, because these fatty acids contribute to enhance the properties of biodiesel (such as density, viscosity, flash point and heating value) and thus improve the quality of biodiesel (Hu et al., 2008). In addition, the cells cultured outdoors achieved higher MUFAs (monounsaturated fatty acids) contents (38.71%) and much lower PUFAs (polyunsaturated fatty acids) content (20.5%) compared to those obtained indoors (MUFAs content of 29.84%, PUFAs content of 35.96%). Moreover, a higher SFAs (saturated fatty acids content) (40.63%) was noted in the cells under outdoor conditions, compared to that (31.24%) obtained under indoor condition. Many previous studies showed that light intensity influences the degree of fatty acid saturation and the components of fatty acids (Ho et al., 2012). High light can alter fatty acid synthesis in many algal cells to increase the contents of SFAs that mainly make up neutral lipids (Fabregas et al., 2004; Hu et al., 2008). In outdoor

Fig. 3. Dry weight, biomass productivity and lmax of Scenedesmus obliquus in six growth phases (G1, G2, G3, G4, G5 and G6) of semi-continuous cultivation outdoors. The culture medium was BG-11 (nitrogen-sufficient). Data are represented as mean ± standard deviation of triplicates.

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Table 5 Specific growth rate, dry weight, biomass productivity, lipid content and productivity of Scenedesmus obliquus in semi-continuous cultivation outdoors.a

a b c d

Cultural phaseb

Specific growth rate lmax (day1)c

Dry weight (g L1)c

Biomass productivity (mg L1 day1)c

Lipid content (%)d

Lipid productivity (mg L1 day1)d

G1/L1 G2/L2 G3/L3 G4/L4 G5/L5 G6/L6

1.35 ± 0.02 1.22 ± 0.03 1.44 ± 0.01 1.11 ± 0.02 1.14 ± 0.04 1.05 ± 0.03

4.32 ± 0.19 4.45 ± 0.17 4.25 ± 0.12 3.96 ± 0.15 3.85 ± 0.13 3.8 ± 0.12

525 ± 24 542 ± 40 513 ± 29 473 ± 19 466 ± 30 452 ± 20

48.8 ± 1.0 49.6 ± 0.8 48.5 ± 1.5 47.9 ± 1.1 46.7 ± 1.2 46.3 ± 1.4

185 ± 28 193 ± 26 178 ± 23 163 ± 25 158 ± 20 151 ± 21

Data are reported as mean ± standard deviation of triplicates. S. obliquus cells were cultured for 61 days including six growth phases (G1, G2, G3, G4, G5 and G6) and lipid accumulation (L1, L2, L3, L4, L5 and L6) phases. lmax, dry weight, and biomass productivity obtained in growth phases (G1, G2, G3, G4, G5 and G6). Lipid content and lipid productivity obtained in lipid accumulation (L1, L2, L3, L4, L5 and L6) phases.

Fig. 4. Dry weight, lipid contents and productivity of Scenedesmus obliquus in six lipid accumulation phases (L1, L2, L3, L4, L5 and L6) of semi-continuous cultivation outdoors. The cells were cultured in nitrogen-free medium. Data are expressed as mean ± standard deviation of triplicates.

cultivation of current experiments, the daily average light intensity was (about 1500 lmol m2 s2) much higher than that (400 lmol m2 s2) indoors. That might be an important reason caused the amounts of SFAs in S. obliquus cells was higher outdoors compared to that (31.24%) obtained indoors. Previous reports showed that SFAs produce a biodiesel with superior oxidative stability and high cetane number (Hu et al., 2008). Therefore, the outdoor cultivation could enhance the oxidative stability of biodiesel produced from S. obliquus. According to above results, the growth rate of S. obliquus dropped a little in outdoor cultivation, but the cells reached higher biomass productivity, lipid content and productivity compared to those obtained under indoor condition. Moreover, the C16/C18 content of S. obliquus cultivated outdoors could still reach a high level (95.54% of total fatty acids). Therefore, S. obliquus is a promising strain for lipids production by outdoor cultivation.

3.4. Culturing S. obliquus by two-phase process in semi-continuous cultivation outdoors An important aspect of commercially successful algal culture is the ability to grow the algae in continuous or semi-continuous culture for long periods (Moheimani and Borowitzka, 2006). That can optimize the use of capital intensive culture systems and also reduce labor costs (Sheen et al., 1998). To evaluate the lipid production potential of S. obliquus under outdoor condition, the cells were grown outdoors in 80 L FPPs under nitrogen sufficient and deficient conditions. The experiments were carried out between Sep. 15 and Nov. 14. S. obliquus cells (initial dry cell weight of 0.122 g L1) were inoculated into 80 L FPPs outdoors with nitrogen sufficient medium and cultured for 8 days (growth phase). Then, most of the cultures were transferred into another 80 L FPPs with nitrogen deficient

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medium for accumulating lipids (7 days of lipid accumulation phase). In addition, fresh nitrogen sufficient medium was added into the remaining cultures for next growth phase. Finally, the cycle of growth phase and lipid accumulation phase were repeated six times (the total cultivation time was 61 days), including 6 growth phases (G1, G2, G3, G4, G5 and G6) and lipid accumulation (L1, L2, L3, L4, L5 and L6) phases.

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outdoor conditions to reach high growth rate and lipid productivity. In addition, stable high lipid production (151193 mg L1 day1) could be achieved by semi-continuous culture of S. obliquus in flat plate photobioreactors outdoors. The results could serve as a foundation for further scale-up cultivation of S. obliquus for biomass and biodiesel production. Acknowledgements

3.4.1. Growth of S. obliquus in semi-continuous cultivation outdoors The changes of dry weight, maximum biomass productivity and lmax (maximum specific growth rate) were shown in Fig. 3. During the experiments, the daily (8:30 am–5:30 pm) temperature variation of medium was between 24 °C and 35 °C, and the daily average light intensity was about 1500 lmol m2 s2 (the highest light intensity was above 2200 lmol m2 s2). S. obliquus cells grew well in all the growth phases (Fig. 3a). During the semicontinuous cultivation outdoors, the biomass productivities obtained in 6 growth phases were between 450 and 550 mg L1 day1, the final dry weights were P3.8 g L1, and the lmax were 1.05–1.44 day1 (Fig. 3b; Table 5). As shown in Table 5, the highest (542 mg L1 day1) biomass productivity (542 mg L1 day1) and dry weight (4.45 g L1) of S. obliquus were both reached in G2 (Sep. 24–Oct. 2), but the highest lmax (1.44 day1) was obtained in G3. In addition, the lowest biomass productivity (452 mg L1 day1), dry weight (3.8 g L1) and lmax (1.05 day1) all occurred in G6 cultivation. The results presented here show that it is feasible to grow S. obliquus in FPPs for long periods under outdoor conditions. 3.4.2. Lipid accumulation of S. obliquus in semi-continuous cultivation outdoors The dry weight, lipid contents productivity of S. obliquus in semi-continuous cultivation was showed in Fig. 4. Under outdoor nitrogen deficient conditions, the lipid contents of cells increased markedly and reached above 46% of dry cell weight on day 8 in all lipid accumulation (L1, L2, L3, L4, L5 and L6) phases of the semi-continuous cultivation (Fig. 4b; Table 5). The results also shows that the lipid contents obtained in each phase are similar to each other, the highest lipid content of 49.6% was obtained in L2 phase. In the end, the cells also achieved high lipid productivities in cultivation were in a range of 151–193 mg L1 day1 (Table 5, Fig. 4c). These results also indicated that a stable high lipid production can be achieved by semi-continuous culture of S. obliquus in FPPs outdoors. Although the scale of photobioreactor tested here is far from that of a commercial facility, it seems clear that the semi-continuous mass cultivation of S. obliquus for producing lipids outdoors is possible. However, it should be noted that some uncertainty issues and uncontrollable factors might influence the growth and lipid accumulation of S. obliquus in semi-continuous cultivation outdoors, such as geography variation, circadian rhythms, seasonal changes, weather conditions, bioreactors, scale of cultivation, pollutions by protozoa, rotifera, other algal species and bacteria etc. (Moheimani and Borowitzka, 2006; Rodolfi et al., 2009; Feng et al., 2011). Therefore, further research is required for the scaling up cultivation, long-term semi-continuous operation, increasing biomass/lipid productivities and optimizing outdoor cultural conditions. 4. Conclusions This study has further demonstrated that S. obliquus is promising specie for biodiesel feedstock production. The cells had the ability to grow at high growth rate and achieve high lipid content of 49.6% outdoors. S. obliquus could also adapt the fluctuating

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