Bioresource Technology 174 (2014) 274–280
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Selection of microalgae for biodiesel production in a scalable outdoor photobioreactor in north China Ling Xia a, Shaoxian Song a, Qiaoning He b,c, Haijian Yang b,c, Chunxiang Hu b,⇑ a
School of Resources and Environmental Engineering, Wuhan University of Technology, Wuhan 430070, China Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China c University of Chinese Academy of Sciences, Beijing 100049, China b
h i g h l i g h t s Eight algal species were analyzed for growth, lipid accumulation and FA profiles. Closed sterile culture and open culture were used for strain selection. Culture for strain characterization was scaling up. 3
Average bio-oil productivity of 22.8 m ha
1
yr1.
S. obtusus XJ-15 showed high capacity for biofuel production in north China.
a r t i c l e
i n f o
Article history: Received 13 August 2014 Received in revised form 27 September 2014 Accepted 1 October 2014 Available online 14 October 2014 Keywords: Desmodesmus Scenedesmus Photobioreactor Scale up Biodiesel
a b s t r a c t The aim of this study was to identify the most promising species as biodiesel feedstock for large-scale cultivation in north China. Eight species of microalgae, selected on the basis of indoor screening, were tested for lipid productivity and the suitability of their fatty acid profiles for biodiesel production under outdoor conditions. Among them, three species Desmodesmus sp. NMX451, Desmodesmus sp. T28-1 and Scenedesmus obtusus XJ-15 were selected for further characterization due to their possessing higher lipid productivities and favorable biodiesel properties. The best strain was S. obtusus XJ-15, with highest biomass productivity of 20.2 g m2 d1 and highest lipid content of 31.7% in a culture of 140 L. S. obtusus XJ-15 was further identified as the best candidate for liquid biofuel production, characterized by average areal growth rate of 23.8 g m2 d1 and stable lipid content of above 31.0% under a scale of 1400 L over a season. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Biodiesel have recently attracted extensive interests as it is carbon-neutral and environment-friendly. Among various sources for biodiesel production, microalgae are considered as the most promising feedstock for the future of biofuel production because they have high photosynthetic efficiency, high growth rate, and can be cultivated on non-arable land (Chisti, 2007). Moreover, the growth of microalgae at the same time will contribute to Greenhouse Gas savings (Wang et al., 2008). In spite of many advances, producing microalgal oil for biodiesel is still too expensive. In fact, the first step in an algal process is to choose the right alga with relevant properties. However, the robust algal growth and high lipid production are reversely related ⇑ Corresponding author. Tel./fax: +86 27 68780866. E-mail address: [email protected] (C. Hu). http://dx.doi.org/10.1016/j.biortech.2014.10.008 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
(Rawat et al., 2013). Certain strains of microalgae, such as Botryococcus braunii, have high lipid storage potential (75% by dry cell weight (DCW)) but this is accompanied by low biomass productivity (Mata et al., 2010; Rawat et al., 2013). Due to this contradiction, lipid productivity showed a combination of biomass productivity and lipid production was considered as the most important selection parameter (Griffiths and Harrison, 2009). The appropriate oil quality, besides its yields, is also key desirable characteristic of algal-based biodiesel industry because it influences the efficiency of biodiesel conversion and its quality (Nascimento et al., 2013; Rawat et al., 2013; Talebi et al., 2013). For example, Nannochloropsis, as one of the most promising sources of oil feedstock for biodiesel production, has high biomass production capacity and relatively high lipid content (Rodolfi et al., 2009); however, being rich in long chain polyunsaturated fatty acids (PUFA) (Doan et al., 2011; Griffiths et al., 2012), which is not desirable for biodiesel properties (e.g., ignition quality and oxidative
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L. Xia et al. / Bioresource Technology 174 (2014) 274–280
stability) (Doan et al., 2011). Thus, it should be analyzed thoroughly. In addition, to minimize costs, photoautotrophic microalgal biodiesel production must rely on freely available sunlight. Qualities generally desirable for outdoor mass culture for microalgal lipid production include resistance to contamination, tolerance to a wide range of environmental conditions (above all temperature and solar radiation changes), rapid CO2 uptake and tolerance to shear force (Griffiths et al., 2012). Various oil-rich microalgae, particularly those belonging to the genera Chlorella and Scenedesmus have been considered as potential sources of renewable energy (Ho et al., 2010; Song et al., 2013). Especially, Scenedesmus have often been used for photosynthetic CO2 reduction combined with biodiesel production (Ho et al., 2010; Toledo-Cervantes et al., 2013). In addition, Desmodesmus, which are identified as thermotolerant genus, have recently drawn many attentions (Pan et al., 2011). Thus, eight microalgae strains in this study belong to Scenedesmus or Desmodesmus were used to examine the capacities of oil production under outdoor conditions. Moreover, not all the oil-rich microalgae can be cultivated under outdoor conditions in certain area. For example, oleaginous microalgae Tetraselmis suecica CS-187 and Chlorella sp. were successfully realized a long-term outdoor cultivation in Victoria, Australia, but Dunaliella tertiolecta CS-175 failed to scale up after many tries (Moheimani, 2012). In this study, an algal screening system was first attempted to expand culture and maintain a large scale cultivation using the selected microalgae in north China. The experiments were all conducted in a greenhouse in Beijing, China using sunlight as energy source. The objective of this study was to determine whether the eight green algal species had the potential to accumulate lipid and select the most promising species for large scale cultivation. Biomass and lipid productivities, lipid profiles and the estimated biodiesel properties were selected as critical factors for the evaluation. The selected robust species were further examined via a cultivation of 140 L in the columns to decide the best strain for biodiesel production. Moreover, this study describes the culture of the selected strain in a larger scale of 1400 L over a period of three months.
et al. (2013, 2014) in the green house in Beijing, China (40°220 N, 116°200 E). For strain selection, the cultures were grown in 5 L Erlenmeyer flask (0.37 m height 0.22 m in diameter) with 3 L medium. BG-11 was prepared from tap water which was filtered through 1 lm polypropylene filters (FTW) or autoclaved distilled water (ADW) and then added with nutrient solutions, respectively for open culture and closed culture. The closed culture was sealed with cotton plug and aerated continuously with sterile filtered air, while open culture sealed with filter paper and aerated with air without any treatment. The closed one kept sterile operation during the whole period of cultivation while the open one did not. To ensure well cell mixing, a 5 cm magnetic stir bar (mixing at 150 rpm) was placed at the middle of the bioreactor chamber for stirring. CO2 in air (0.03–5%, v/v) was supplied to each bioreactor using an air compressor through the pipage during the daytime. The following large scale trials using the selected microalgae were carried out in 140 L bioreactor, which composed with two connected 70 L hanging column bags (1.80 m height 0.22 m in diameter). CO2 in air (0.03–5%, v/v) was supplied to each bioreactor using an air compressor through the pipage during the daytime. For all the trials, the initial cell concentration was 0.1 g L1, and culture media was BG-11 with nitrogen source of only 0.2 g L1 urea because urea is significantly less expensive and exhibited more favorable effect on algal growth in outdoor cultures (Xia et al., 2013, 2014). The average day time high and night time low air temperatures during the tested period were 39 °C and 23 °C. 2.3. Analytical procedures 2.3.1. Biomass measurement The biomass of 500 mL culture cells was harvested by centrifugation, then the wet cell mass was frozen overnight at 70 °C and freeze-dried at 54 °C under a vacuum (Alpha 1-2 LD plus, Christ). The biomass concentration (BC, mg L1) of the tested microalgae was determined by measuring optical density of 680 nm (OD680) via an ultraviolet photospectrometer and using the following equations:
BC ðD:abundans T12Þ ¼ 320 OD680 ðR2 ¼ 0:996Þ
ð1Þ
2. Methods
BC ðDesmodesmus sp: T28-1Þ ¼ 322 OD680 ðR2 ¼ 0:999Þ
ð2Þ
2.1. Organisms and culture
BC ðDesmodesmus sp: NMX451Þ ¼ 230 OD680 ðR2 ¼ 0:998Þ
ð3Þ
The eight microalgal species (Desmodesmus abundans T12, Desmodesmus sp. T28-1, Desmodesmus sp. NMX451, Desmodesmus intermedius HB12-2, Desmodesmus obtusus XJ-36, Scenedesmus pectinatus var XJ-1, Scenedesmus obtusus XJ-15, Scenedesmus obtusus XJ-19) used in this study, gifted by prof. Xu Xudong of Institute of Hydrobiology in China, was selected after a laboratory screening. Among these, D. abundans T12 and Desmodesmus sp. T28-1 were isolated from one of the largest Chinese freshwater lake (Lake Taihu). Desmodesmus sp. NMX451 and S. obtusus XJ-19 were isolated from Inner Mongolia of China. The rest of the species were isolated from reservoirs or ponds in Hubei province in China. Stock cultures for all the strains were grown in modified BG-11 medium containing 300 mg NaNO3, 30 mg K2HPO4, 36 mg CaCl22H2O, 6 mg ammonium citrate monohydrate, 6 mg ammonium ferric citrate, 1 mg EDTA, 2.86 lg H3BO3, 1.81 lg MnCl24H2O, 0.222 lg ZnSO47H2O, 0.39 lg NaMoO45H2O, 0.079 lg CuSO45H2O, 0.050 lg CoCl26H2O in 1 L sterile distilled water.
BC ðD:intermedius HB12-2Þ ¼ 213 OD680 ðR2 ¼ 0:998Þ
ð4Þ
BC ðD:obtusus XJ 36Þ ¼ 201 OD680 ðR2 ¼ 0:997Þ
ð5Þ
BC ðS:pectinatus v ar XJ-1Þ ¼ 277 OD680 ðR2 ¼ 0:998Þ
ð6Þ
BC ðS:obtusus XJ-15Þ ¼ 229 OD680 ðR2 ¼ 0:996Þ
ð7Þ
2.2. Experimental setup All experiments were conducted in bioreactors (5 L flask bioreactor or 140 L airlift bag columns) as described previously by Xia
BC ðS:obtusus XJ-19Þ ¼ 368 OD680 ðR2 ¼ 0:999Þ
ð8Þ 1
The volumetric biomass productivity (VBP, mg L culated according to the Eq. (9):
VBP ¼ ðB2 B1 Þ=T
1
d
) was cal-
ð9Þ
where B2 and B1 represents the dry weight biomass density at the time T (days) and at the start of the experiment, respectively. The areal biomass productivity (ABP, g m2 d1) was calculated according to the Eq. (10). The calculation was based on VBP and the floor area (required for columns and additional area required for operational convenience such as empty space between reactors as well as ground area to avoid shading effect), which is 0.26 m2 for each column.
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ABP ¼ ðVBP 70Þ=ð0:26 1000Þ
L. Xia et al. / Bioresource Technology 174 (2014) 274–280
ð10Þ
2.3.2. Lipid analysis The total lipid was gravimetrically quantified after extraction using a Soxhlet’s extractor with chloroform/methanol (1/2, v/v) as solvent and incubated at 90 °C for 4 h. Lipid class separation was performed by silica gel column chromatography. Solvent sequences were as following: 6 volumes of chloroform to collect the neutral lipid factions and 6 volumes of methanol for polar lipids. The lipid productivity (LP, mg L1 d1) was determined according to the Eq. (11):
LP ¼ BP LC
ð11Þ
where LC denotes the total lipid content (w/dry weight biomass, %) at time T. Biodiesel was determined as fatty acid methyl esters (FAME) after acidic transesterification of lipids. In brief, 100 mg of lipid sample was suspended in 1 M H2SO4–methanol (2 mL) in a vial. The vial was flushed with nitrogen to ensure an inert atmosphere before sealing and heated at 100 °C for 1 h in a water bathe. After methylation, purified water (0.20 mL) and n-hexane (0.60 mL) were added, the mixture centrifuged and the top hexane layer contained FAME were collected. 1 lL FAME in hexane was injected into gas chromatograph mass spectrometry (GC–MS; Thermo Scientific ITQ 700™, USA) for analysis. All parameters of the FAME were derived from the calibration curves generated from the FAME standard mix (Supelco 37 component FAME mix, Sigma–Aldrich). Generally, the direct measurement of critical biodiesel quality requires large amounts of oil and specialized devices, which are not always available. Using the predictive models based on FA composition makes the estimation of fuel property easy and quick, which is of importance in biodiesel-based strain selection (Nascimento et al., 2013; Talebi et al., 2013; Song et al., 2014). The most important feedstock-related properties of biodiesel were found to correlate with composition of the FA, providing also linear best-fit curves (Hoekman et al., 2012; Nascimento et al., 2013; Talebi et al., 2013). The accuracy of these empirical equations in estimating the quality of biodiesel has been previously shown (Ramos et al., 2009; Hoekman et al., 2012). Moreover, measured values and calculated values by these equations in green algae C. protothecoides (Xu et al., 2006), Neochloris oleabundans (Gouveia et al., 2009) and S. obliquus (Abd El Baky et al., 2012) were compared prior to this study, and results indicated that the values from equations of Song et al. (2014) were very close to the measured values. Thus, the biodiesel property in this study, the degree of unsaturation (DU), kinematic viscosity (KS), specific gravity (SG), cloud point (CP), iodine value (IV), cetane number (CN) and higher heating value (HHV) were determined by empirical equations from FA composition as described by Song et al. (2014), while cold filter plugging point (CFPP) by Nascimento et al. (2013). 2.3.3. Mass balance estimation A simple mass balance from a culture scale of 1400 L is applied according to Khoo et al. (2011) as follows: ? From the average total lipid content of 31.5% in algal biomass and 51.7% triacylglycerol (TAG) content in total lipid, 1 kg of dry microalgae biomass can produce a theoretical maximum of 0.16 kg of TAG. ? From 1 kg TAG produced, assuming 90% is converted to biodiesel. Therefore the total amount of dry algal biomass required to produce 1 kg biodiesel = 6.8 kg dry biomass.
3. Result and discussion 3.1. Strain selection 3.1.1. Growth and lipid accumulation properties of eight green algae In any algal process, species selection is a key decision influencing choice of location, reactor design, culture conditions, harvesting method and product range (Griffiths et al., 2012). The trials of strain selection were performed using autoclaved distilled water (ADW) and filtered tap water (FTW). The former was conducted in the closed sterile culture and the later open culture. The closed one was used to test the ability of algal cells to tolerating environmental changes, especially in light intensity and temperature. The open one was to further examine the resilience ability of cells to contamination. Eight freshwater microalgal strains (Desmodesmus and Scenedesmus) were evaluated for their lipid production potential by evaluating their biomass productivity and lipid content under the above cultivation conditions (Table 1). After 15 days of batch cultivation, the cultures reached the highest biomass productivity, and harvested for lipid analysis. Both average biomass productivity and lipid productivity of the eight microalgal strains under ADW (BP, 200.3 mg L1 d1; LP, 45.4 mg L1 d1) were higher than that under FTW (BP, 163.7 mg L1 d1; LP, 38.1 mg L1 d1). In addition, the lipid contents in all the strains were also higher under ADW than that under FTW, except for Desmodesmus sp. T28-1 and D. obtusus XJ-36. These results indicate that water quality is an important factor affecting the production capacity of microalgae, and should be considered in the strain selection. The highest lipid content occurred in S. obtusus XJ-15 under ADW; the value (31.31%) is higher than the literature data in Scenedesmus indoors (Griffiths and Harrison, 2009; Nascimento et al., 2013). The lipid content was still increasing after 15 days and may have reached higher values if cultivation had been continued. Moreover, biomass and lipid productivities of Desmodesmus sp. T28-1 and D. abundans T12 were much higher under FTW. The results suggest that Desmodesmus sp. T28-1 and D. abundans T12 showed excellent weatherability and anti-contamination properties. Lipid productivity has been suggested as the most appropriate kinetic parameter for the comparison of the species for biodiesel production (Griffiths and Harrison, 2009). Though in the same class, the 10 microalgal species showed different abilities to accumulate lipid, which indicated the fundamental importance of algal species screening and evaluation for lipid production. In general, the high lipid accumulation and robust biomass production were mutually exclusive (Rawat et al., 2013). But under outdoor conditions, considering the weather factors, S. obtusus XJ-15 had the highest biomass productivity and, at the same time the highest lipid content. The result suggest that strain selection under outdoor conditions was very important. The maximum lipid productivity was reached by S. obtusus XJ-15 under ADW, and by Desmodesmus sp. T28-1 under AFW, followed by D. abundans T12. Thus, S. obtusus XJ-15, Desmodesmus sp. T28-1 and D. abundans T12 presented the highest lipid productivity and emerged from the screening as the best candidates for algal oil production.
3.1.2. Fatty acid profiling and estimated biodiesel properties of eight green algae GC profiles for algal strains under FTW used in this study are tabulated in Table 2. As seen, the C16-C18 FA, which is the most common feedstocks suitable for biodiesel, possessed over 97% of total FA in the eight green microalgae tested. The predominant FAMEs found in all the algal oils were C16:0 and C18:1, with the
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L. Xia et al. / Bioresource Technology 174 (2014) 274–280 Table 1 Biomass and lipid productivity of 8 microalgal strains cultivated in 5 L flasks outdoors. Strains
D. abundans T12 Desmodesmus sp. T28-1 Desmodesmus sp. NMX-451 D. intermedius HB12-2 D. obtusus XJ-36 S. obtusus XJ-15 S. obtusus XJ-19 S. pectinatus var XJ-1
Biomass concentration (g L1)
Lipid content (%)
Biomass productivity (mg L1 d1)
Lipid productivity (mg L1 d1)
ADW
FTW
ADW
FTW
ADW
FTW
ADW
FTW
2.6 2.7 2.4 2.6 2.6 3.6 2.4 2.8
3.0 3.2 2.0 1.9 1.6 3.3 3.0 1.8
25.1 24.6 25.5 24.6 23.1 31.3 22.7 23.2
23.2 26.6 23.2 23.3 24.1 24.1 18.5 22.6
171.5 180.0 160.1 169.7 170.3 238.4 160.0 183.5
199.0 210.9 134.0 125.9 104.8 222.7 193.3 119.2
43.3 44.3 40.8 41.8 39.3 74.6 36.4 42.5
46.2 56.1 31.1 29.3 25.5 53.7 35.8 26.9
ADW, autoclaved distilled water; FTW, filtered tap water.
Table 2 Fatty acid profile (%) of biodiesel from eight green algal oils in 5 L flasks outdoors using tap water. D. abundans T12
Desmodesmus sp. T28-1
Desmodesmus sp. NMX-451
D. intermedius HB12-2
D. obtusus XJ-36
S. obtusus XJ-19
S. obtusus XJ-15
S. pectinatus var XJ-1
Saturated fatty acids (% of total FAME)
C14:0 C16:0 C18:0 C20:0 Subtotal
0.94 3.30 11.01 1.32 16.57
0.24 46.06 6.18 1.20 53.68
0.18 56.71 8.20 0.74 65.83
0.49 39.33 6.01 0.61 46.44
0.68 43.98 6.09 ND 50.75
1.19 46.25 3.07 ND 50.51
0.56 39.82 6.11 ND 46.49
0.33 38.64 3.54 0.36 50.51
Monoenoic fatty acids (% of total FAME)
C14:1 C16:1 C18:1 Subtotal
0.10 3.12 57.48 60.70
0.01 3.98 38.41 42.40
0.03 2.2 29.01 31.24
0.01 3.55 40.56 44.12
0.11 0.04 28.86 29.01
0.43 6.57 30.87 37.87
0.10 4.14 29.12 33.36
0.02 0.14 38.7 37.45
Polyenoic fatty acids (% of total FAME)
C18:2 C18:3 C18:4 Subtotal
9.18 13.56 ND 22.74
2.46 1.37 0.09 3.92
1.66 1.19 0.08 2.93
5.66 3.49 0.30 9.45
9.81 9.48 0.93 20.22
5.41 5.84 0.37 11.62
9.74 9.52 0.87 20.13
9.86 7.85 0.57 6.20
C16–C18 (% of total FAME)
C16– C18
97.64
98.46
98.97
98.59
98.28
98.02
98.47
98.72
ND: not detected.
individual amounts varying significantly. For example, Desmodesmus sp. NMX451 possessed the highest content of C16:0 (56.71% DCW), while D. abundans T12 contained rather low content of 3.30%. Relatively, D. abundans T12 possessed the highest content of C18:1 (57.48% DCW), while D. obtusus XJ-36 contained rather low content of 28.86%. The peaks for Palmitic acid (C16:0) and oleic acid (C18:1) in Desmodesmus and Scenedesmus were consistent with the observations by Nascimento et al. (2013) and Song et al. (2014). In addition, highly unsaturated FA, C20–C24 with 3–6 double bonds, appear to be more commonly occurring in the FA of algal oils but barely detected in the eight microalgae (Doan et al., 2011;). The consensus view is that the most favorable biodiesel would have rather low levels of polyunsaturated (PUFA) and low levels of saturated FA (SFA) to decrease oxidative stability and low temperature property (Knothe, 2009). However, the rather high lipid content of SFA (C16:0) in the tested strains would resulted in cold flow problems. Nevertheless, the feedstock rich in SFA have higher CN than fuels produced from less saturated feedstock (Knothe, 2009; Hoekman et al., 2012). Moreover, SFA and PUFA can easily be converted to monounsaturated FA (MUFA) when changing the culture conditions in green algae (Griffiths et al., 2012). In addition, oxidative stability is determined not only by FAME composition, but also by the age of the biodiesel and the conditions under which it has been stored (Hoekman et al., 2012). In addition, the carbon– carbon double bond orientation is also important for the trans configuration is more stable than cis (Hoekman et al., 2012). Although some strains in this study, such as S. obtusus XJ-15, had a high content of PUFA (20%), its oxidative stability required further investigation.
Table 3 presents the most important properties of biodiesel predicted based on FA profiles for the 8 microalgae strains studied as well as for oil-rich microalgae Nannochloropsis sp. and traditional crops rape and soybean. All the microalgae tested satisfied the criterion set by the Chinese National Standards (Table 3). Moreover, according to the two most common quality standards for biodiesel, ASTM D6751 in the US and EN 14214 in Europe, the values of kinematic viscosity, specific gravity, cetane number and iodine number of the eight candidates satisfied the specifications, except for D. abundans T12. The oxidative instability factor IV had a high value beyond the standard in D. abundans T12 as well as in Nannochloropsis sp. and soybean (Table 3). However, biodiesel property of D. abundans T12 can be improved when subjected to high salt or high alkaline (Xia et al., 2014). The CN is a prime indicator of biodiesel quality, and related to the ignition delay time and combustion quality. The higher the CN is, the better is in its ignition properties. CN in all the tested species showed a higher value than that in Nannochloropsis sp. and soybean (Table 3), which could help ensure better cold start properties and minimize the formation of white smoke. Similar findings also have been observed in other studies (Griffiths et al., 2012; Talebi et al., 2013). There are no definite specifications of cloud point (CP) and cold filter plugging point (CFPP), due to the different climate condition in the United States and Europe (Knothe, 2011). Nonetheless, all of the species examined met the winter specification, even for a relatively warm country such as Spain (10 °C; Ramos et al., 2009) or South Africa (3 °C; Stansell et al., 2011). It is noticeable that Desmodesmus sp. NMX51 showed the best biodiesel property with the highest CN value and the lowest IV
– Report
3.2. Growth of three selected microalgae in a 140 L airlift column bioreactor
39.73 16.09
40.03 16.00
40.02 14.95
41.19 15.32
– 5
– Report
The environmental factors including light intensity, temperature and pH value fluctuated during the cultivation. Of the above parameters, light intensity was affected by the weather most. When the weather was clear, the light intensity on the surface of the column was as high as 500 lmol photons m2 s1. And once in rainy days, the illuminations were close to zero. The haze in north China was another important factor affecting the illumination. The temperature changed with the illumination but the degree of variation was smaller than the light intensity. Temperature of the medium was controlled at a value under 32 °C with cold system in the green house at daytime, and the temperature at night was at 20–28 °C. The pH value was regulated by the aeration of CO2. The CO2 concentration in the atmosphere aerated was depending on the weather. When it is sunny, the concentration of CO2 was as high as 5%, and when cloudy or rainy, under 1%. The pH value of the medium during cultivation was controlled at 8–10, which allowed a robust growth of microalgal cells and also could help resist contamination such as zooplankton and microorganisms because of the alkaline environment (Bartley et al., 2014). The growth and lipid accumulation properties of the selected elite microalgae Desmodesmus sp. NMX451, Desmodesmus sp. T28-1 and S. obtusus XJ-15 in the 140 L bioreactors were summarized in Table 4. The trials were conducted for 13 days, but the effective illumination time only lasted for 9 days. And during the foggy and wet days with little illumination, the cells barely grew. Although all of the three species studied survived during the weather changes and recorded a high yield, the biomass yield decreased by about 25% (Table 4). Therefore, the yields from certain strain cultivated in certain area are very important and necessary, which provided data support to the true potential for the algal biofuel production. The actual areal biomass productivity obtained in both Desmodesmus sp. T28-1 and S. obtusus XJ-15 was above 20 g m2 d1. Moreover, the lipid content in S. obtusus XJ-15 without lipid induction phase was as high as 31.0% of DCW, which met the commercial production of biodiesel production (Chisti, 2007). Therefore, S. obtusus XJ-15 showed the best biodiesel potential and selected for larger scale investigation.
39.71 14.09 39.21 13.43
39.96 15.97
– – 128 125.08 101.32 101.68 88.86 88.43 67.13
98.63
0.88 8.29 58.40 0.87 8.31 60.31
0.88 8.28 57.49
0.88 8.29 58.36
0.88 8.28 57.21
0.88 8.28 57.25
0.86 0.18 52.80
– – 49
0.85–0.90 – Min 47
– – Min 49
– 1.96.0
– 3.5– 5.0 – – Min 51 Max 120 – Report – 1.9–6.0 1.44 4.20 1.51 4.25 0.84 4.67 0.85 4.67 0.68 4.78 0.81 4.70 0.67 4.78 0.38 4.96
S. obtusus XJ-15 S. obtusus XJ-19 D. obtusus XJ-36 D. intermedius HB12-2 Desmodesmus sp. NMX-451
value. Therefore, the traits of contamination toleration, high lipid productivity and good biodiesel property potential make S. obtusus XJ-15, Desmodesmus sp. T28-1 and Desmodesmus sp. NMX51 suitable candidates for bioenergy production and selected for further analysis.
39.45 12.19 40.64 11.96
Based on Doan et al. (2011). Ramos et al. (2009). a
b
77.06
0.88 8.30 59.42 0.88 8.25 54.89
127.58
Iodine number (g I2 100 g1) HHV (MJ/kg) CFPP (°C)
0.52 4.88 1.20 4.45
Avg. unsaturation Kinematic viscosity 40 °C (mm2 s1) Specific gravity (kg L1) Cloud point (°C) Cetane number
Desmodesmus sp. T28-1
3.3. Cultivating S. obtusus XJ-15 under a scale of 1400 L
D. abundans T12
Table 3 PredictedEstimated properties of biodiesel based on fatty acid profiles from eight green algal oils in 5 L flasks outdoors using tap water.
S. pectinatus var XJ-1
Nannochloropsis sp.a
Soybeanb
ASTMD6751
GB/T 208282007
L. Xia et al. / Bioresource Technology 174 (2014) 274–280
EN 14214
278
To further evaluate the potential of the selected elite microalgae for biofuel production, S. obtusus XJ-15 was inoculated into a row of connected columns in an area of 5.4 m2 with a scale of 1400 L. Table 5 presents the biomass and lipid productivities after three runs of batch cultivation using untreated tap water during June to September, 2013. In the first and the third run, algae recorded a volumetric biomass productivity of about 100 mg L1 d1, corresponding to an areal biomass productivity of over 26 g m2 d1. However, the second run recorded a lower productivity of 68.6 mg L1 d1. This is largely due to the relatively low light intensity in haze days during late July and early August (Table 5). It was evident from the results that weather factors especially light intensity directly influences the outdoor yield of microalgae (Moheimani, 2012; Hindersin et al., 2014). Therefore, compensative artificial light source is necessary and important when the solar light intensity decreased to a value that could not
279
L. Xia et al. / Bioresource Technology 174 (2014) 274–280 Table 4 Biomass and lipid productivities of S. obtusus XJ-15, Desmodesmus sp. NMX451 and Desmodesmus sp. T28-1 cultivated in a 140 L photobioreactor. Biomass concentration (g L1)
Desmodesmus sp. T28–1 Desmodesmus sp. NMX451 S. obtusus XJ-15 a b
0.8 ± 0.1 0.9 ± 0.0 0.9 ± 0.0
Volumetric biomass productivity (mg L1 d1)
Areal biomass productivity (g m2 d1)
Actuala
Theoreticalb
Actuala
Theoreticalb
75.4 ± 7.6 68.7 ± 3.2 75.6 ± 3.6
97.0 ± 9.8 103.0 ± 10.4 98.1 ± 9.4
20.1 ± 2.0 18.3 ± 1.1 20.2 ± 1.0
25.9 ± 2.6 27.5 ± 2.0 26.1 ± 2.5
Lipid content (%)
Lipid productivity (mg L1 d1)
21.7 ± 0.7 27.06 ± 0.8 31.7 ± 1.5
21.0 ± 0.2 27.87 ± 1.2 31.0 ± 2.8
Including foggy, cloudy and rainy days. Clear days with effective illumination.
Table 5 Biomass and lipid productivities of S. obtusus XJ-15 in a scale of 1400 L. Run
1 2 3
Biomass concentration (g L1)
Lipid content (%)
Biomass productivity Volumetric (mg L1 d1)
Areal (g m2 d1)
1.1 ± 0.3 0.8 ± 0.1 0.9 ± 0.0
31.7 ± 1.5 31.4 ± 0.8 31.4 ± 0.8
98.1 ± 9.4 68.6 ± 11.7 100.8 ± 4.9
26.1 ± 2.5 18.3 ± 3.1 26.9 ± 1.3
support microalgal growth. However, the average biomass productivity of the three runs reached 23.8 g m2 d1, which is higher than most of the oil-rich microalgae recorded in the literatures (Chen et al., 2011; Nurra et al., 2014). Moreover, the lipid content in the three runs maintained stably at above 31%, with an average content of 31.5%. This lipid content was much higher than most of the records in the outdoor cultures, indicating a lipid content of 15.6% for Botryococcus braunii (Bazaes et al., 2012), 19–32% for Chlorella sp (Moheimani, 2012, 2013), 34.7% for N. oculata and N. salina (Quinn et al., 2012), less than 30% for S. acutus (Doria et al., 2011), and 24–30% for Tetraselmis suecica (Moheimani, 2013). In addition, the lipid productivity averaged at 28.1 mg L1 d1, peaked at 31.7 mg L1 d1. The lipid production achievable by S. obtusus XJ-15 is equivalent to 22.8 m3 ha1 yr1 for a 9-month production period. And this value was two times higher against 10.7 m3 ha1 yr1 achieved by Nannochloropsis (Quinn et al., 2012). Regarding the biomass productivity and the biomass balance estimation, it can be predicted that the strain S. obtusus XJ-15 has the capability to produce 10,600 L ha1 yr1 of biodiesel. As for the rest cold winter days of the year in north China, further work on exploring and culturing other candidates which can grow and accumulate useful components in this period is needed to reduce the total cost for biodiesel production. Thus, S. obtusus XJ-15 showed an outstanding capacity for oil production, and it is suitable for microalgae cultured in north China. 4. Conclusions Eight microalgae were investigated for their potential for biodiesel production under outdoor conditions. In addition to comparing the two key characteristics (lipid productivity and fatty acid profile) for promising microalgae species, this work highlighted the importance of tolerances to water quality and environment factors in screening microalgal strains. Through examination, S. obtusus XJ-15 was finally selected and cultivated in a scale of 1400 L through three turns. Both high lipid content (31%) and high lipid productivity (22.8 m3 ha1 yr1) were achieved. This study assesses the importance and helps to fill the gap of the microalgae cultivated in north China. Acknowledgements This work was supported by National 863 program (2013AA065804) and Program of Sinopec, international partner
Lipid productivity (mg L1 d1)
Light intensity (lmol photons m2 s1)
Temperature (°C)
31.0 ± 2.8 21.6 ± 3.6 31.7 ± 1.7
665.5 372.0 438.1
27.3 26.9 28.4
program of innovation team (Chinese Academy of Sciences), Platform construction of oleaginous microalgae (Institute of Hydrobiology, CAS of China).
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Selection of microalgae for biodiesel production in a scalable outdoor photobioreactor in north China Ling Xia a, Shaoxian Song a, Qiaoning He b,c, Haijian Yang b,c, Chunxiang Hu b,⇑ a
School of Resources and Environmental Engineering, Wuhan University of Technology, Wuhan 430070, China Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China c University of Chinese Academy of Sciences, Beijing 100049, China b
h i g h l i g h t s Eight algal species were analyzed for growth, lipid accumulation and FA profiles. Closed sterile culture and open culture were used for strain selection. Culture for strain characterization was scaling up. 3
Average bio-oil productivity of 22.8 m ha
1
yr1.
S. obtusus XJ-15 showed high capacity for biofuel production in north China.
a r t i c l e
i n f o
Article history: Received 13 August 2014 Received in revised form 27 September 2014 Accepted 1 October 2014 Available online 14 October 2014 Keywords: Desmodesmus Scenedesmus Photobioreactor Scale up Biodiesel
a b s t r a c t The aim of this study was to identify the most promising species as biodiesel feedstock for large-scale cultivation in north China. Eight species of microalgae, selected on the basis of indoor screening, were tested for lipid productivity and the suitability of their fatty acid profiles for biodiesel production under outdoor conditions. Among them, three species Desmodesmus sp. NMX451, Desmodesmus sp. T28-1 and Scenedesmus obtusus XJ-15 were selected for further characterization due to their possessing higher lipid productivities and favorable biodiesel properties. The best strain was S. obtusus XJ-15, with highest biomass productivity of 20.2 g m2 d1 and highest lipid content of 31.7% in a culture of 140 L. S. obtusus XJ-15 was further identified as the best candidate for liquid biofuel production, characterized by average areal growth rate of 23.8 g m2 d1 and stable lipid content of above 31.0% under a scale of 1400 L over a season. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Biodiesel have recently attracted extensive interests as it is carbon-neutral and environment-friendly. Among various sources for biodiesel production, microalgae are considered as the most promising feedstock for the future of biofuel production because they have high photosynthetic efficiency, high growth rate, and can be cultivated on non-arable land (Chisti, 2007). Moreover, the growth of microalgae at the same time will contribute to Greenhouse Gas savings (Wang et al., 2008). In spite of many advances, producing microalgal oil for biodiesel is still too expensive. In fact, the first step in an algal process is to choose the right alga with relevant properties. However, the robust algal growth and high lipid production are reversely related ⇑ Corresponding author. Tel./fax: +86 27 68780866. E-mail address: [email protected] (C. Hu). http://dx.doi.org/10.1016/j.biortech.2014.10.008 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
(Rawat et al., 2013). Certain strains of microalgae, such as Botryococcus braunii, have high lipid storage potential (75% by dry cell weight (DCW)) but this is accompanied by low biomass productivity (Mata et al., 2010; Rawat et al., 2013). Due to this contradiction, lipid productivity showed a combination of biomass productivity and lipid production was considered as the most important selection parameter (Griffiths and Harrison, 2009). The appropriate oil quality, besides its yields, is also key desirable characteristic of algal-based biodiesel industry because it influences the efficiency of biodiesel conversion and its quality (Nascimento et al., 2013; Rawat et al., 2013; Talebi et al., 2013). For example, Nannochloropsis, as one of the most promising sources of oil feedstock for biodiesel production, has high biomass production capacity and relatively high lipid content (Rodolfi et al., 2009); however, being rich in long chain polyunsaturated fatty acids (PUFA) (Doan et al., 2011; Griffiths et al., 2012), which is not desirable for biodiesel properties (e.g., ignition quality and oxidative
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L. Xia et al. / Bioresource Technology 174 (2014) 274–280
stability) (Doan et al., 2011). Thus, it should be analyzed thoroughly. In addition, to minimize costs, photoautotrophic microalgal biodiesel production must rely on freely available sunlight. Qualities generally desirable for outdoor mass culture for microalgal lipid production include resistance to contamination, tolerance to a wide range of environmental conditions (above all temperature and solar radiation changes), rapid CO2 uptake and tolerance to shear force (Griffiths et al., 2012). Various oil-rich microalgae, particularly those belonging to the genera Chlorella and Scenedesmus have been considered as potential sources of renewable energy (Ho et al., 2010; Song et al., 2013). Especially, Scenedesmus have often been used for photosynthetic CO2 reduction combined with biodiesel production (Ho et al., 2010; Toledo-Cervantes et al., 2013). In addition, Desmodesmus, which are identified as thermotolerant genus, have recently drawn many attentions (Pan et al., 2011). Thus, eight microalgae strains in this study belong to Scenedesmus or Desmodesmus were used to examine the capacities of oil production under outdoor conditions. Moreover, not all the oil-rich microalgae can be cultivated under outdoor conditions in certain area. For example, oleaginous microalgae Tetraselmis suecica CS-187 and Chlorella sp. were successfully realized a long-term outdoor cultivation in Victoria, Australia, but Dunaliella tertiolecta CS-175 failed to scale up after many tries (Moheimani, 2012). In this study, an algal screening system was first attempted to expand culture and maintain a large scale cultivation using the selected microalgae in north China. The experiments were all conducted in a greenhouse in Beijing, China using sunlight as energy source. The objective of this study was to determine whether the eight green algal species had the potential to accumulate lipid and select the most promising species for large scale cultivation. Biomass and lipid productivities, lipid profiles and the estimated biodiesel properties were selected as critical factors for the evaluation. The selected robust species were further examined via a cultivation of 140 L in the columns to decide the best strain for biodiesel production. Moreover, this study describes the culture of the selected strain in a larger scale of 1400 L over a period of three months.
et al. (2013, 2014) in the green house in Beijing, China (40°220 N, 116°200 E). For strain selection, the cultures were grown in 5 L Erlenmeyer flask (0.37 m height 0.22 m in diameter) with 3 L medium. BG-11 was prepared from tap water which was filtered through 1 lm polypropylene filters (FTW) or autoclaved distilled water (ADW) and then added with nutrient solutions, respectively for open culture and closed culture. The closed culture was sealed with cotton plug and aerated continuously with sterile filtered air, while open culture sealed with filter paper and aerated with air without any treatment. The closed one kept sterile operation during the whole period of cultivation while the open one did not. To ensure well cell mixing, a 5 cm magnetic stir bar (mixing at 150 rpm) was placed at the middle of the bioreactor chamber for stirring. CO2 in air (0.03–5%, v/v) was supplied to each bioreactor using an air compressor through the pipage during the daytime. The following large scale trials using the selected microalgae were carried out in 140 L bioreactor, which composed with two connected 70 L hanging column bags (1.80 m height 0.22 m in diameter). CO2 in air (0.03–5%, v/v) was supplied to each bioreactor using an air compressor through the pipage during the daytime. For all the trials, the initial cell concentration was 0.1 g L1, and culture media was BG-11 with nitrogen source of only 0.2 g L1 urea because urea is significantly less expensive and exhibited more favorable effect on algal growth in outdoor cultures (Xia et al., 2013, 2014). The average day time high and night time low air temperatures during the tested period were 39 °C and 23 °C. 2.3. Analytical procedures 2.3.1. Biomass measurement The biomass of 500 mL culture cells was harvested by centrifugation, then the wet cell mass was frozen overnight at 70 °C and freeze-dried at 54 °C under a vacuum (Alpha 1-2 LD plus, Christ). The biomass concentration (BC, mg L1) of the tested microalgae was determined by measuring optical density of 680 nm (OD680) via an ultraviolet photospectrometer and using the following equations:
BC ðD:abundans T12Þ ¼ 320 OD680 ðR2 ¼ 0:996Þ
ð1Þ
2. Methods
BC ðDesmodesmus sp: T28-1Þ ¼ 322 OD680 ðR2 ¼ 0:999Þ
ð2Þ
2.1. Organisms and culture
BC ðDesmodesmus sp: NMX451Þ ¼ 230 OD680 ðR2 ¼ 0:998Þ
ð3Þ
The eight microalgal species (Desmodesmus abundans T12, Desmodesmus sp. T28-1, Desmodesmus sp. NMX451, Desmodesmus intermedius HB12-2, Desmodesmus obtusus XJ-36, Scenedesmus pectinatus var XJ-1, Scenedesmus obtusus XJ-15, Scenedesmus obtusus XJ-19) used in this study, gifted by prof. Xu Xudong of Institute of Hydrobiology in China, was selected after a laboratory screening. Among these, D. abundans T12 and Desmodesmus sp. T28-1 were isolated from one of the largest Chinese freshwater lake (Lake Taihu). Desmodesmus sp. NMX451 and S. obtusus XJ-19 were isolated from Inner Mongolia of China. The rest of the species were isolated from reservoirs or ponds in Hubei province in China. Stock cultures for all the strains were grown in modified BG-11 medium containing 300 mg NaNO3, 30 mg K2HPO4, 36 mg CaCl22H2O, 6 mg ammonium citrate monohydrate, 6 mg ammonium ferric citrate, 1 mg EDTA, 2.86 lg H3BO3, 1.81 lg MnCl24H2O, 0.222 lg ZnSO47H2O, 0.39 lg NaMoO45H2O, 0.079 lg CuSO45H2O, 0.050 lg CoCl26H2O in 1 L sterile distilled water.
BC ðD:intermedius HB12-2Þ ¼ 213 OD680 ðR2 ¼ 0:998Þ
ð4Þ
BC ðD:obtusus XJ 36Þ ¼ 201 OD680 ðR2 ¼ 0:997Þ
ð5Þ
BC ðS:pectinatus v ar XJ-1Þ ¼ 277 OD680 ðR2 ¼ 0:998Þ
ð6Þ
BC ðS:obtusus XJ-15Þ ¼ 229 OD680 ðR2 ¼ 0:996Þ
ð7Þ
2.2. Experimental setup All experiments were conducted in bioreactors (5 L flask bioreactor or 140 L airlift bag columns) as described previously by Xia
BC ðS:obtusus XJ-19Þ ¼ 368 OD680 ðR2 ¼ 0:999Þ
ð8Þ 1
The volumetric biomass productivity (VBP, mg L culated according to the Eq. (9):
VBP ¼ ðB2 B1 Þ=T
1
d
) was cal-
ð9Þ
where B2 and B1 represents the dry weight biomass density at the time T (days) and at the start of the experiment, respectively. The areal biomass productivity (ABP, g m2 d1) was calculated according to the Eq. (10). The calculation was based on VBP and the floor area (required for columns and additional area required for operational convenience such as empty space between reactors as well as ground area to avoid shading effect), which is 0.26 m2 for each column.
276
ABP ¼ ðVBP 70Þ=ð0:26 1000Þ
L. Xia et al. / Bioresource Technology 174 (2014) 274–280
ð10Þ
2.3.2. Lipid analysis The total lipid was gravimetrically quantified after extraction using a Soxhlet’s extractor with chloroform/methanol (1/2, v/v) as solvent and incubated at 90 °C for 4 h. Lipid class separation was performed by silica gel column chromatography. Solvent sequences were as following: 6 volumes of chloroform to collect the neutral lipid factions and 6 volumes of methanol for polar lipids. The lipid productivity (LP, mg L1 d1) was determined according to the Eq. (11):
LP ¼ BP LC
ð11Þ
where LC denotes the total lipid content (w/dry weight biomass, %) at time T. Biodiesel was determined as fatty acid methyl esters (FAME) after acidic transesterification of lipids. In brief, 100 mg of lipid sample was suspended in 1 M H2SO4–methanol (2 mL) in a vial. The vial was flushed with nitrogen to ensure an inert atmosphere before sealing and heated at 100 °C for 1 h in a water bathe. After methylation, purified water (0.20 mL) and n-hexane (0.60 mL) were added, the mixture centrifuged and the top hexane layer contained FAME were collected. 1 lL FAME in hexane was injected into gas chromatograph mass spectrometry (GC–MS; Thermo Scientific ITQ 700™, USA) for analysis. All parameters of the FAME were derived from the calibration curves generated from the FAME standard mix (Supelco 37 component FAME mix, Sigma–Aldrich). Generally, the direct measurement of critical biodiesel quality requires large amounts of oil and specialized devices, which are not always available. Using the predictive models based on FA composition makes the estimation of fuel property easy and quick, which is of importance in biodiesel-based strain selection (Nascimento et al., 2013; Talebi et al., 2013; Song et al., 2014). The most important feedstock-related properties of biodiesel were found to correlate with composition of the FA, providing also linear best-fit curves (Hoekman et al., 2012; Nascimento et al., 2013; Talebi et al., 2013). The accuracy of these empirical equations in estimating the quality of biodiesel has been previously shown (Ramos et al., 2009; Hoekman et al., 2012). Moreover, measured values and calculated values by these equations in green algae C. protothecoides (Xu et al., 2006), Neochloris oleabundans (Gouveia et al., 2009) and S. obliquus (Abd El Baky et al., 2012) were compared prior to this study, and results indicated that the values from equations of Song et al. (2014) were very close to the measured values. Thus, the biodiesel property in this study, the degree of unsaturation (DU), kinematic viscosity (KS), specific gravity (SG), cloud point (CP), iodine value (IV), cetane number (CN) and higher heating value (HHV) were determined by empirical equations from FA composition as described by Song et al. (2014), while cold filter plugging point (CFPP) by Nascimento et al. (2013). 2.3.3. Mass balance estimation A simple mass balance from a culture scale of 1400 L is applied according to Khoo et al. (2011) as follows: ? From the average total lipid content of 31.5% in algal biomass and 51.7% triacylglycerol (TAG) content in total lipid, 1 kg of dry microalgae biomass can produce a theoretical maximum of 0.16 kg of TAG. ? From 1 kg TAG produced, assuming 90% is converted to biodiesel. Therefore the total amount of dry algal biomass required to produce 1 kg biodiesel = 6.8 kg dry biomass.
3. Result and discussion 3.1. Strain selection 3.1.1. Growth and lipid accumulation properties of eight green algae In any algal process, species selection is a key decision influencing choice of location, reactor design, culture conditions, harvesting method and product range (Griffiths et al., 2012). The trials of strain selection were performed using autoclaved distilled water (ADW) and filtered tap water (FTW). The former was conducted in the closed sterile culture and the later open culture. The closed one was used to test the ability of algal cells to tolerating environmental changes, especially in light intensity and temperature. The open one was to further examine the resilience ability of cells to contamination. Eight freshwater microalgal strains (Desmodesmus and Scenedesmus) were evaluated for their lipid production potential by evaluating their biomass productivity and lipid content under the above cultivation conditions (Table 1). After 15 days of batch cultivation, the cultures reached the highest biomass productivity, and harvested for lipid analysis. Both average biomass productivity and lipid productivity of the eight microalgal strains under ADW (BP, 200.3 mg L1 d1; LP, 45.4 mg L1 d1) were higher than that under FTW (BP, 163.7 mg L1 d1; LP, 38.1 mg L1 d1). In addition, the lipid contents in all the strains were also higher under ADW than that under FTW, except for Desmodesmus sp. T28-1 and D. obtusus XJ-36. These results indicate that water quality is an important factor affecting the production capacity of microalgae, and should be considered in the strain selection. The highest lipid content occurred in S. obtusus XJ-15 under ADW; the value (31.31%) is higher than the literature data in Scenedesmus indoors (Griffiths and Harrison, 2009; Nascimento et al., 2013). The lipid content was still increasing after 15 days and may have reached higher values if cultivation had been continued. Moreover, biomass and lipid productivities of Desmodesmus sp. T28-1 and D. abundans T12 were much higher under FTW. The results suggest that Desmodesmus sp. T28-1 and D. abundans T12 showed excellent weatherability and anti-contamination properties. Lipid productivity has been suggested as the most appropriate kinetic parameter for the comparison of the species for biodiesel production (Griffiths and Harrison, 2009). Though in the same class, the 10 microalgal species showed different abilities to accumulate lipid, which indicated the fundamental importance of algal species screening and evaluation for lipid production. In general, the high lipid accumulation and robust biomass production were mutually exclusive (Rawat et al., 2013). But under outdoor conditions, considering the weather factors, S. obtusus XJ-15 had the highest biomass productivity and, at the same time the highest lipid content. The result suggest that strain selection under outdoor conditions was very important. The maximum lipid productivity was reached by S. obtusus XJ-15 under ADW, and by Desmodesmus sp. T28-1 under AFW, followed by D. abundans T12. Thus, S. obtusus XJ-15, Desmodesmus sp. T28-1 and D. abundans T12 presented the highest lipid productivity and emerged from the screening as the best candidates for algal oil production.
3.1.2. Fatty acid profiling and estimated biodiesel properties of eight green algae GC profiles for algal strains under FTW used in this study are tabulated in Table 2. As seen, the C16-C18 FA, which is the most common feedstocks suitable for biodiesel, possessed over 97% of total FA in the eight green microalgae tested. The predominant FAMEs found in all the algal oils were C16:0 and C18:1, with the
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L. Xia et al. / Bioresource Technology 174 (2014) 274–280 Table 1 Biomass and lipid productivity of 8 microalgal strains cultivated in 5 L flasks outdoors. Strains
D. abundans T12 Desmodesmus sp. T28-1 Desmodesmus sp. NMX-451 D. intermedius HB12-2 D. obtusus XJ-36 S. obtusus XJ-15 S. obtusus XJ-19 S. pectinatus var XJ-1
Biomass concentration (g L1)
Lipid content (%)
Biomass productivity (mg L1 d1)
Lipid productivity (mg L1 d1)
ADW
FTW
ADW
FTW
ADW
FTW
ADW
FTW
2.6 2.7 2.4 2.6 2.6 3.6 2.4 2.8
3.0 3.2 2.0 1.9 1.6 3.3 3.0 1.8
25.1 24.6 25.5 24.6 23.1 31.3 22.7 23.2
23.2 26.6 23.2 23.3 24.1 24.1 18.5 22.6
171.5 180.0 160.1 169.7 170.3 238.4 160.0 183.5
199.0 210.9 134.0 125.9 104.8 222.7 193.3 119.2
43.3 44.3 40.8 41.8 39.3 74.6 36.4 42.5
46.2 56.1 31.1 29.3 25.5 53.7 35.8 26.9
ADW, autoclaved distilled water; FTW, filtered tap water.
Table 2 Fatty acid profile (%) of biodiesel from eight green algal oils in 5 L flasks outdoors using tap water. D. abundans T12
Desmodesmus sp. T28-1
Desmodesmus sp. NMX-451
D. intermedius HB12-2
D. obtusus XJ-36
S. obtusus XJ-19
S. obtusus XJ-15
S. pectinatus var XJ-1
Saturated fatty acids (% of total FAME)
C14:0 C16:0 C18:0 C20:0 Subtotal
0.94 3.30 11.01 1.32 16.57
0.24 46.06 6.18 1.20 53.68
0.18 56.71 8.20 0.74 65.83
0.49 39.33 6.01 0.61 46.44
0.68 43.98 6.09 ND 50.75
1.19 46.25 3.07 ND 50.51
0.56 39.82 6.11 ND 46.49
0.33 38.64 3.54 0.36 50.51
Monoenoic fatty acids (% of total FAME)
C14:1 C16:1 C18:1 Subtotal
0.10 3.12 57.48 60.70
0.01 3.98 38.41 42.40
0.03 2.2 29.01 31.24
0.01 3.55 40.56 44.12
0.11 0.04 28.86 29.01
0.43 6.57 30.87 37.87
0.10 4.14 29.12 33.36
0.02 0.14 38.7 37.45
Polyenoic fatty acids (% of total FAME)
C18:2 C18:3 C18:4 Subtotal
9.18 13.56 ND 22.74
2.46 1.37 0.09 3.92
1.66 1.19 0.08 2.93
5.66 3.49 0.30 9.45
9.81 9.48 0.93 20.22
5.41 5.84 0.37 11.62
9.74 9.52 0.87 20.13
9.86 7.85 0.57 6.20
C16–C18 (% of total FAME)
C16– C18
97.64
98.46
98.97
98.59
98.28
98.02
98.47
98.72
ND: not detected.
individual amounts varying significantly. For example, Desmodesmus sp. NMX451 possessed the highest content of C16:0 (56.71% DCW), while D. abundans T12 contained rather low content of 3.30%. Relatively, D. abundans T12 possessed the highest content of C18:1 (57.48% DCW), while D. obtusus XJ-36 contained rather low content of 28.86%. The peaks for Palmitic acid (C16:0) and oleic acid (C18:1) in Desmodesmus and Scenedesmus were consistent with the observations by Nascimento et al. (2013) and Song et al. (2014). In addition, highly unsaturated FA, C20–C24 with 3–6 double bonds, appear to be more commonly occurring in the FA of algal oils but barely detected in the eight microalgae (Doan et al., 2011;). The consensus view is that the most favorable biodiesel would have rather low levels of polyunsaturated (PUFA) and low levels of saturated FA (SFA) to decrease oxidative stability and low temperature property (Knothe, 2009). However, the rather high lipid content of SFA (C16:0) in the tested strains would resulted in cold flow problems. Nevertheless, the feedstock rich in SFA have higher CN than fuels produced from less saturated feedstock (Knothe, 2009; Hoekman et al., 2012). Moreover, SFA and PUFA can easily be converted to monounsaturated FA (MUFA) when changing the culture conditions in green algae (Griffiths et al., 2012). In addition, oxidative stability is determined not only by FAME composition, but also by the age of the biodiesel and the conditions under which it has been stored (Hoekman et al., 2012). In addition, the carbon– carbon double bond orientation is also important for the trans configuration is more stable than cis (Hoekman et al., 2012). Although some strains in this study, such as S. obtusus XJ-15, had a high content of PUFA (20%), its oxidative stability required further investigation.
Table 3 presents the most important properties of biodiesel predicted based on FA profiles for the 8 microalgae strains studied as well as for oil-rich microalgae Nannochloropsis sp. and traditional crops rape and soybean. All the microalgae tested satisfied the criterion set by the Chinese National Standards (Table 3). Moreover, according to the two most common quality standards for biodiesel, ASTM D6751 in the US and EN 14214 in Europe, the values of kinematic viscosity, specific gravity, cetane number and iodine number of the eight candidates satisfied the specifications, except for D. abundans T12. The oxidative instability factor IV had a high value beyond the standard in D. abundans T12 as well as in Nannochloropsis sp. and soybean (Table 3). However, biodiesel property of D. abundans T12 can be improved when subjected to high salt or high alkaline (Xia et al., 2014). The CN is a prime indicator of biodiesel quality, and related to the ignition delay time and combustion quality. The higher the CN is, the better is in its ignition properties. CN in all the tested species showed a higher value than that in Nannochloropsis sp. and soybean (Table 3), which could help ensure better cold start properties and minimize the formation of white smoke. Similar findings also have been observed in other studies (Griffiths et al., 2012; Talebi et al., 2013). There are no definite specifications of cloud point (CP) and cold filter plugging point (CFPP), due to the different climate condition in the United States and Europe (Knothe, 2011). Nonetheless, all of the species examined met the winter specification, even for a relatively warm country such as Spain (10 °C; Ramos et al., 2009) or South Africa (3 °C; Stansell et al., 2011). It is noticeable that Desmodesmus sp. NMX51 showed the best biodiesel property with the highest CN value and the lowest IV
– Report
3.2. Growth of three selected microalgae in a 140 L airlift column bioreactor
39.73 16.09
40.03 16.00
40.02 14.95
41.19 15.32
– 5
– Report
The environmental factors including light intensity, temperature and pH value fluctuated during the cultivation. Of the above parameters, light intensity was affected by the weather most. When the weather was clear, the light intensity on the surface of the column was as high as 500 lmol photons m2 s1. And once in rainy days, the illuminations were close to zero. The haze in north China was another important factor affecting the illumination. The temperature changed with the illumination but the degree of variation was smaller than the light intensity. Temperature of the medium was controlled at a value under 32 °C with cold system in the green house at daytime, and the temperature at night was at 20–28 °C. The pH value was regulated by the aeration of CO2. The CO2 concentration in the atmosphere aerated was depending on the weather. When it is sunny, the concentration of CO2 was as high as 5%, and when cloudy or rainy, under 1%. The pH value of the medium during cultivation was controlled at 8–10, which allowed a robust growth of microalgal cells and also could help resist contamination such as zooplankton and microorganisms because of the alkaline environment (Bartley et al., 2014). The growth and lipid accumulation properties of the selected elite microalgae Desmodesmus sp. NMX451, Desmodesmus sp. T28-1 and S. obtusus XJ-15 in the 140 L bioreactors were summarized in Table 4. The trials were conducted for 13 days, but the effective illumination time only lasted for 9 days. And during the foggy and wet days with little illumination, the cells barely grew. Although all of the three species studied survived during the weather changes and recorded a high yield, the biomass yield decreased by about 25% (Table 4). Therefore, the yields from certain strain cultivated in certain area are very important and necessary, which provided data support to the true potential for the algal biofuel production. The actual areal biomass productivity obtained in both Desmodesmus sp. T28-1 and S. obtusus XJ-15 was above 20 g m2 d1. Moreover, the lipid content in S. obtusus XJ-15 without lipid induction phase was as high as 31.0% of DCW, which met the commercial production of biodiesel production (Chisti, 2007). Therefore, S. obtusus XJ-15 showed the best biodiesel potential and selected for larger scale investigation.
39.71 14.09 39.21 13.43
39.96 15.97
– – 128 125.08 101.32 101.68 88.86 88.43 67.13
98.63
0.88 8.29 58.40 0.87 8.31 60.31
0.88 8.28 57.49
0.88 8.29 58.36
0.88 8.28 57.21
0.88 8.28 57.25
0.86 0.18 52.80
– – 49
0.85–0.90 – Min 47
– – Min 49
– 1.96.0
– 3.5– 5.0 – – Min 51 Max 120 – Report – 1.9–6.0 1.44 4.20 1.51 4.25 0.84 4.67 0.85 4.67 0.68 4.78 0.81 4.70 0.67 4.78 0.38 4.96
S. obtusus XJ-15 S. obtusus XJ-19 D. obtusus XJ-36 D. intermedius HB12-2 Desmodesmus sp. NMX-451
value. Therefore, the traits of contamination toleration, high lipid productivity and good biodiesel property potential make S. obtusus XJ-15, Desmodesmus sp. T28-1 and Desmodesmus sp. NMX51 suitable candidates for bioenergy production and selected for further analysis.
39.45 12.19 40.64 11.96
Based on Doan et al. (2011). Ramos et al. (2009). a
b
77.06
0.88 8.30 59.42 0.88 8.25 54.89
127.58
Iodine number (g I2 100 g1) HHV (MJ/kg) CFPP (°C)
0.52 4.88 1.20 4.45
Avg. unsaturation Kinematic viscosity 40 °C (mm2 s1) Specific gravity (kg L1) Cloud point (°C) Cetane number
Desmodesmus sp. T28-1
3.3. Cultivating S. obtusus XJ-15 under a scale of 1400 L
D. abundans T12
Table 3 PredictedEstimated properties of biodiesel based on fatty acid profiles from eight green algal oils in 5 L flasks outdoors using tap water.
S. pectinatus var XJ-1
Nannochloropsis sp.a
Soybeanb
ASTMD6751
GB/T 208282007
L. Xia et al. / Bioresource Technology 174 (2014) 274–280
EN 14214
278
To further evaluate the potential of the selected elite microalgae for biofuel production, S. obtusus XJ-15 was inoculated into a row of connected columns in an area of 5.4 m2 with a scale of 1400 L. Table 5 presents the biomass and lipid productivities after three runs of batch cultivation using untreated tap water during June to September, 2013. In the first and the third run, algae recorded a volumetric biomass productivity of about 100 mg L1 d1, corresponding to an areal biomass productivity of over 26 g m2 d1. However, the second run recorded a lower productivity of 68.6 mg L1 d1. This is largely due to the relatively low light intensity in haze days during late July and early August (Table 5). It was evident from the results that weather factors especially light intensity directly influences the outdoor yield of microalgae (Moheimani, 2012; Hindersin et al., 2014). Therefore, compensative artificial light source is necessary and important when the solar light intensity decreased to a value that could not
279
L. Xia et al. / Bioresource Technology 174 (2014) 274–280 Table 4 Biomass and lipid productivities of S. obtusus XJ-15, Desmodesmus sp. NMX451 and Desmodesmus sp. T28-1 cultivated in a 140 L photobioreactor. Biomass concentration (g L1)
Desmodesmus sp. T28–1 Desmodesmus sp. NMX451 S. obtusus XJ-15 a b
0.8 ± 0.1 0.9 ± 0.0 0.9 ± 0.0
Volumetric biomass productivity (mg L1 d1)
Areal biomass productivity (g m2 d1)
Actuala
Theoreticalb
Actuala
Theoreticalb
75.4 ± 7.6 68.7 ± 3.2 75.6 ± 3.6
97.0 ± 9.8 103.0 ± 10.4 98.1 ± 9.4
20.1 ± 2.0 18.3 ± 1.1 20.2 ± 1.0
25.9 ± 2.6 27.5 ± 2.0 26.1 ± 2.5
Lipid content (%)
Lipid productivity (mg L1 d1)
21.7 ± 0.7 27.06 ± 0.8 31.7 ± 1.5
21.0 ± 0.2 27.87 ± 1.2 31.0 ± 2.8
Including foggy, cloudy and rainy days. Clear days with effective illumination.
Table 5 Biomass and lipid productivities of S. obtusus XJ-15 in a scale of 1400 L. Run
1 2 3
Biomass concentration (g L1)
Lipid content (%)
Biomass productivity Volumetric (mg L1 d1)
Areal (g m2 d1)
1.1 ± 0.3 0.8 ± 0.1 0.9 ± 0.0
31.7 ± 1.5 31.4 ± 0.8 31.4 ± 0.8
98.1 ± 9.4 68.6 ± 11.7 100.8 ± 4.9
26.1 ± 2.5 18.3 ± 3.1 26.9 ± 1.3
support microalgal growth. However, the average biomass productivity of the three runs reached 23.8 g m2 d1, which is higher than most of the oil-rich microalgae recorded in the literatures (Chen et al., 2011; Nurra et al., 2014). Moreover, the lipid content in the three runs maintained stably at above 31%, with an average content of 31.5%. This lipid content was much higher than most of the records in the outdoor cultures, indicating a lipid content of 15.6% for Botryococcus braunii (Bazaes et al., 2012), 19–32% for Chlorella sp (Moheimani, 2012, 2013), 34.7% for N. oculata and N. salina (Quinn et al., 2012), less than 30% for S. acutus (Doria et al., 2011), and 24–30% for Tetraselmis suecica (Moheimani, 2013). In addition, the lipid productivity averaged at 28.1 mg L1 d1, peaked at 31.7 mg L1 d1. The lipid production achievable by S. obtusus XJ-15 is equivalent to 22.8 m3 ha1 yr1 for a 9-month production period. And this value was two times higher against 10.7 m3 ha1 yr1 achieved by Nannochloropsis (Quinn et al., 2012). Regarding the biomass productivity and the biomass balance estimation, it can be predicted that the strain S. obtusus XJ-15 has the capability to produce 10,600 L ha1 yr1 of biodiesel. As for the rest cold winter days of the year in north China, further work on exploring and culturing other candidates which can grow and accumulate useful components in this period is needed to reduce the total cost for biodiesel production. Thus, S. obtusus XJ-15 showed an outstanding capacity for oil production, and it is suitable for microalgae cultured in north China. 4. Conclusions Eight microalgae were investigated for their potential for biodiesel production under outdoor conditions. In addition to comparing the two key characteristics (lipid productivity and fatty acid profile) for promising microalgae species, this work highlighted the importance of tolerances to water quality and environment factors in screening microalgal strains. Through examination, S. obtusus XJ-15 was finally selected and cultivated in a scale of 1400 L through three turns. Both high lipid content (31%) and high lipid productivity (22.8 m3 ha1 yr1) were achieved. This study assesses the importance and helps to fill the gap of the microalgae cultivated in north China. Acknowledgements This work was supported by National 863 program (2013AA065804) and Program of Sinopec, international partner
Lipid productivity (mg L1 d1)
Light intensity (lmol photons m2 s1)
Temperature (°C)
31.0 ± 2.8 21.6 ± 3.6 31.7 ± 1.7
665.5 372.0 438.1
27.3 26.9 28.4
program of innovation team (Chinese Academy of Sciences), Platform construction of oleaginous microalgae (Institute of Hydrobiology, CAS of China).
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