Bioresource Technology 180 (2015) 79–87
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Sufficient utilization of natural fluctuating light intensity is an effective approach of promoting lipid productivity in oleaginous microalgal cultivation outdoors Qiaoning He a,b, Haijian Yang a,b, Liangliang Xu a,b, Ling Xia a, Chunxiang Hu a,⇑ a b
Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China University of Chinese Academy of Sciences, Beijing 100049, China
h i g h l i g h t s Six microalgae were identified and analyzed for growth and lipid accumulation. 0.25 g L
1
urea was optimal concentration for culture outdoors.
Lipid accumulation under natural fluctuating light intensities was investigated. Lipid yield and neutral lipid content were enhanced by high fluctuating light intensity. The reduction in glycolipids contributed to NL accumulation under HFI.
a r t i c l e
i n f o
Article history: Received 31 October 2014 Received in revised form 23 December 2014 Accepted 24 December 2014 Available online 31 December 2014 Keywords: Microalgae Fluctuating light intensity Lipid productivity Neutral lipid Glycolipid
a b s t r a c t The effects of fluctuating intensity of solar radiation on biomass and lipid in oleaginous microalgae are important. However, this topic has not been the subject of studies for a long time. In this study, four oleaginous microalgae from semi-arid areas were screened and cultivated outdoors under different fluctuating intensities. Results showed that the highest lipid productivities and neutral lipid (NL) contents occurred under high fluctuating intensity (HFI), in which 13–20% of the increased NL came from glycolipid transformation without phospholipid conversion. Chlorella sp. L1 and Monoraphidium dybowskii Y2 obtained from biological soil crusts in desert had the largest biomass (137.13, 106.61 mg L1 d1) and lipid yields (35.06, 32.45 mg L1 d1) under HFI. The highest areal lipid productivities of 9.06 and 8.95 g m2 d1 and better biodiesel quality were observed under HFI. Accordingly, sufficiently adopting fluctuating light intensity outdoors to culture microalgae was an economic and effective approach. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Given the rapid depletion of fossil fuels and serious environmental problems caused by the extensive use of fossil fuels resources, exploring and employing alternative and environmentally friendly energy resources are receiving considerable attention (William and Laurens, 2010). Alternative energy resources have also become an important strategic direction of global energy structure. Microalgae with various advantages are currently regarded as one of the effective and exceedingly optimistic alternative energy resources (Hu et al., 2008; Griffiths and Harrison, 2009; Siaut et al., 2011). However, cultivating microalgae at large-scale remains to be a problem (Rodolfi et al., 2009), particularly the lipid ⇑ Corresponding author. Tel./fax: +86 27 68780866. E-mail address: [email protected] (C. Hu). http://dx.doi.org/10.1016/j.biortech.2014.12.088 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
content decreases with increasing cultivation (Yang et al., 2014). Lipid is biologically considered an energy storage of microalgae; its production generally increases as a result of exposure to stressful environment, such as nitrogen, phosphorus, and iron deficiency, high salinity, and high pH etc. (Courchesne et al., 2009). Although the lipid content is enhanced, biomass is usually low and lipid productivity is constrained under such condition. Light is the fundamental driving force in the growth of photoautrophic microalgae. In particular, light significantly affects microalgal growth and lipid accumulation (Solovchenko et al., 2008; Liu et al., 2012). Among the effects of light, fluctuations in light intensity are one of the primary factors that influence the generation of high biomass and lipid content in microalgae (Wahidin et al., 2013). Therefore, the sufficient and effective utilization of sunlight during outdoor cultivation deemed extremely potential. However, previous studies presented conflicting results. Some observations
80
Q. He et al. / Bioresource Technology 180 (2015) 79–87
noted that low light intensity results in the accumulation of lipids and/or triacylglycerols (TAG) (Breuer et al., 2013). By contrast, other studies specified that lipid content can be enhanced by high light intensity (Li et al., 2012; Liu et al., 2012), particularly neutral lipids (NLs) (Sharma et al., 2012; Gwak et al., 2014), and low light essentially increases polar lipids (Hu et al., 2008). Outdoor cultures are subjected daily to cyclic changes in dramatic fluctuations of light intensity. In addition to weather conditions and diurnal cycle, the capability of harvesting light and self-shading in microalgae and the reactor translucent influence lipid accumulation in mass cultivation (Pulz et al., 2001; Simionato et al., 2013). Culture productivity is invariably controlled by the availability of light, particularly when the scale of photobioreactor increases. In fact, more people realize the challenge between cost and productivity in microalgae biodiesel. Thus, according to the local natural light resource, maximizing areal lipid productivity should be a parameter that is better associated for industrializing microalgae biofuels. Investigating the influence of natural light fluctuating intensity on the cultivation of oleaginous microalgae is rarely tested outdoors with photobioreactors; thus, it is still exceedingly relevant to be thoroughly explored. Given the above conditions, this research intended to realize the following objectives: (1) identify the strains of microalgae isolated from the topsoil of semi-arid areas through morphological features and 18S rDNA sequences; (2) examine their potential in oilproducing; (3) optimize nitrogen concentrations in the form of low-cost urea outdoors; and (4) determine the effect of natural fluctuating light intensity on growth, lipid content, and lipid classes at both 5 L and 140 L scales to obtain the highest lipid productivity with better biodiesel quality in unit area by reasonable arrangement of photobioreactors. 2. Methods 2.1. Strains The six microalgae used in this study were isolated from the topsoil of semi-arid areas in the northwest of China. Among them, Chlorella sp. L1 and Monoraphidium dybowskii Y2 were obtained from the biological soil crusts of Tengger desert in Ningxia (37°280 N, 105°000 E); M. dybowskii Y1, M. dybowskii D, Chlorella sp. X, and Nannochloris sp. L2 were obtained from the Loess Plateau in Lanzhou City of Gansu (36°020 N, 103°490 E). All microalgae were stored with BG11 medium in FACHB-Collection (Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China). 2.2. Experimental design 2.2.1. Identification Six strains of microalgae were initially identified by morphology under a microscope and were further determined with 18S rDNA sequences. The total genomic DNA was extracted from algal cells with a DNA isolation kit (TianGen Biotech (Bei Jing) Co., Ltd, China). Polymerase chain reaction (PCR) was performed with general primers 18S-1 (50 -tggttgatcctgccagtagtc–30 and 18S-2 (50 -tgatccttctgcaggttcacc-30 ) to amplify 18S rDNA gene. The PCR products were excised from agarose gel, recovered with the DNA isolation kit, and were then sequenced. Finally phylogenetic analyses were conducted using the NCBI Gene Bank database. 2.2.2. Preliminary screen indoors Six strains of microalgae were cultivated with 400 mL of BG-11 medium in 500 mL Erlenmeyer flasks. The initial OD was 0.3. Cool white fluorescent tubes acted as light source to provide the light
Fig. 1. Maximum-likelihood tree of six microalgae inferred from 18S rDNA gene sequence (strains obtained during this study are underlined). Bootstrap values are shown at the internal nodes for neighbor joining (5000 replications), maximum parsimony (1000 replications) and maximum likelihood (100 replications), respectively, if the node is supported by at least two bootstrap values of 50% or above. Branch lengths correspond to evolutionary distances. A distance of 2 is indicated by the scale.
intensity of 100 lmol photons m2 s1, and the temperature was maintained at 25 ± 1 °C with an air conditioner. Filtered air was supplied to flasks by using an air compressor. 2.2.3. Cultivation outdoors The candidate strains were selected based on their lipid productivity. Scale-up experiments were performed during summer in the bioreactors (5-L flask and 140-L photobioreactor), which were placed inside a green house in Beijing, China (40°220 N, 116°200 E). Aeration in 5-L flasks was at a flow rate of 4 L min/L, and the reactor was stirred with a 5 cm magnetic stir bar (mixing at 150 rpm) at the middle of the reactor. The 140-L photobioreactor was composed of two connected 70-L polyvinylchloride hanging bags (22 cm diameter, 180 cm height) (Dalian Huixin Titanium Equipment Development, Co., Ltd) (Xia et al., 2013). The spatial layout was equipped with 80 photobioreactors in an area of 42 m2. Aeration with 1.8% CO2 was provided to the photobioreactors by using an air compressor as carbon source at daytime and as pure air at night. The temperature in the greenhouse was controlled by an air conditioner at a constant range between 25 °C and 28 °C. 2.2.3.1. Optimization of urea concentration in 5-L flasks outdoors. The BG-11 medium was used outdoors, but the form of nitrogen source in the culture was substituted by urea, which was optimized in 5-L flasks outdoors. The urea concentrations were set at 0, 0.1, 0.25, and 0.5 g/L. The screened four oleaginous microalgae were cultivated with 3.5 L of modified BG11 medium in 5-L flasks at different urea concentrations to induce lipid accumulation. The microalgae were harvested when the cultures reached the late exponential phase. 2.2.3.2. Experiments on natural fluctuating light intensity. Four oleaginous microalgae were operated in 5-L flasks and 140-L photobioreactors with the optimal 0.25 g/L urea concentration. Three different natural fluctuating light intensity levels were provided to examine the effects of natural fluctuating light intensity. In
81
Q. He et al. / Bioresource Technology 180 (2015) 79–87 Table 1 Biomass and lipid production of six microalgae. Strains 1
Biomass (g L ) Biomass productivity (mg L1 d1) Lipid content (%, w/w) Lipid productivity (mg L1 d1)
M. dybowskii D
M. dybowskii Y1
M. dybowskii Y2
Chlorella sp. L1
Chlorella sp. X
Nannochloris sp. L2
1.89 ± 0.08 125.79 ± 5.46 33.09 ± 1.98 41.64 ± 3.33
1.79 ± 0.06 121.16 ± 4.87 33.07 ± 2.61 40.01 ± 2.30
2.36 ± 0.10 157.20 ± 6.79 34.63 ± 1.27 54.39 ± 0.38
2.67 ± 0.08 178.03 ± 5.55 26.92 ± 1.39 47.87 ± 0.95
2.48 ± 0.05 165.63 ± 3.01 19.01 ± 3.21 34.53 ± 0.44
2.54 ± 0.13 169.28 ± 0.90 18.52 ± 1.31 31.92 ± 2.97
Data are the means and standard deviation of three independent experiments, the same below.
Fig. 2. Effect of different urea concentrations on biomass and lipid accumulation in four oleaginous microalgae: (A) Chlorella sp. L1; (B) M. dybowskii Y1; (C) M. dybowskii Y2; (D) M. dybowskii D in 5-L flasks outdoors. BP: biomass productivity; LP: lipid productivity.
particular, these light intensity levels were divided into high fluctuating intensity (HFI), middle fluctuating intensity (MFI, about 60% of HFI) and low fluctuating intensity (LFI, about 30% of HFI). Placing the 5-L flasks on a platform of three different layers and in 140-L photobioreactor, directions, and inclinations were remarkably important in absorbing light, particularly via dislocation arrangement and shading to satisfy the requirements. Irradiance and temperature were measured with a detection system, which recorded the data every 10 min of an average value from 6:00 to 18:00 (Fig. 6). The maximum light intensity in daytime was fluctuated from 990 lmol photons m2 s1 to 1486 lmol photons m2 s1. The average light intensity of every level was changed by the weather, but the same level was uniform in the two types of bioreactors. All experiments were conducted in triplicate from June to August 2013. 2.3. Analytical procedures 2.3.1. Biomass measurement Cells density was determined by measuring optical density at 680 nm (OD680). The following below equations describe the
relationships between dry weight (DW, g/L) and the OD680 values of six strains of microalgae. DW = 0.329 OD680, DW = 0.266 OD680, DW = 0.172 OD680, DW = 0.226 OD680, DW = 0.188 OD680, DW = 0.317 OD680,
R2 = 0.947 R2 = 0.986 R2 = 0.994 R2 = 0.984 R2 = 0.989 R2 = 0.996
(Chlorella sp. L1) (Chlorella sp. X) (M. dybowskii Y1) (M. dybowskii Y2) (M. dybowskii D) (Nannochloris sp. L2)
The microalgae were then harvested by centrifugation and were lyophilized with a vacuum freeze dryer (Alpha 1-2 LD plus, Christ). 2.3.2. Lipid analysis Lipid content: Total lipid was extracted using a Soxhlet reflux extractor with chloroform/methanol (2/1, v/v) that ranged from 50 mg to 100 mg of lyophilized algae, and was then gravimetrically quantified. Lipid classes: Solid phase extraction (SPE) was utilized to separate lipid extracts into three fractions, namely, neutral lipids
82
Q. He et al. / Bioresource Technology 180 (2015) 79–87
Fig. 3. Effect of different fluctuating light intensities on growth and lipid content in four oleaginous microalgae: (A) Chlorella sp. L1; (B) M. dybowskii Y1; (C) M. dybowskii Y2; (D) M. dybowskii D in 5-L flasks outdoors.
(NL), glycolipids (GL) and phospholipids (PL). In particular, SPE was conducted with a column (20 mm 200 mm) containing 4 g of silica cartridges. The extracted lipids (approximately 100 mg) were dissolved in 400 lL of chloroform and were fractionated by using the following solvent systems: neutral lipids with chloroform (six column volumes), glycolipids with acetone: methanol (9:1, v/v) (four column volumes) and phospholipids with methanol (four column volumes). The eluted lipid fractions were then dried under nitrogen at 40 °C, and the mass of each fraction was recorded. Fatty acids (FA) composition: Biodiesel was determined as fatty acid methyl esters (FAMEs) after acidic transesterification of lipids. The fatty acid components of extracted microalgal oil were analyzed by gas chromatograph mass spectrometry (GC–MS; Thermo Scientific ITQ 700, USA) equipped with a flame ionization detector (FID) and a fused silica capillary column (60 m 0.25 mm 0.25 lm; Agilent Technologies, USA). The injector and detector temperatures were maintained at 270 °C and 280 °C, respectively, with an oven temperature gradient of 50 °C to 170 °C at 40 °C min1 after a 1 min hold time at 50 °C, then with an oven temperature gradient of 170 °C to 210 °C at 18 °C min1 after a 1 min hold for 1 min. All parameters of the FAMEs were derived from the calibration curves generated from the FAME standard mix (Supelco 37 component FAME mix, Sigma–Aldrich, USA).
2.3.3. Statistical analysis All values were expressed as the mean ± standard deviation. The data were analyzed by two-way ANOVA using the SPSS statistical software (version 19.0). p < 0.05 was considered to have a statistically significant difference.
3. Results and discussion 3.1. Identification of microalgae The six strains of microalgae isolated from the topsoil of semiarid areas were all identified as green algae by morphological characterization. The 18S rDNA sequences of these strains were then further compared. The results revealed the affiliation of strains L1 and X to Chlorella sp., strains D, Y1, and Y2 to M. dybowskii, and strain L2 to Nannochloris sp. (Fig. 1). 3.2. Strain selection indoors The findings on six microalgae cultured indoors (Table 1) demonstrated that the highest biomass productivity was 178.03 mg L1 d1 in Chlorella sp. L1, followed by Nannochloris sp. L2, Chlorella sp. X, and three M. dybowskii strains. The highest lipid content was observed in M. dybowskii Y2 at 34.63%. The best lipid producer was the strain that possessed the best combination of biomass productivity and lipid content; thus, lipid productivity was the most reliable indicator for oleaginous microalgae. (Rodolfi et al., 2009; Griffiths and Harrison, 2009). M. dybowskii Y2 with maximum lipid productivity (54.39 mg L1 d1), followed by Chlorella sp. L1 (47.87 mg L1 d1), M. dybowskii D (41.64 mg L1 d1), and M. dybowskii Y1 (40.01 mg L1 d1), which all had lipid productivity of more than 40 mg L1 d1, were selected as the potential feedstock for the subsequent scale-up experiments. 3.3. Optimization of urea concentrations outdoors Urea is considered a good choice for large-scale production because of its low cost (Matsudo et al., 2009). Moreover, this
83
Q. He et al. / Bioresource Technology 180 (2015) 79–87
Fig. 4. Effect of different fluctuating light intensities on growth in four oleaginous microalgae: (A) Chlorella sp. L1; (B) M. dybowskii Y1; (C) M. dybowskii Y2; (D) M. dybowskii D in 140-L photobioreactor outdoors.
Table 2 Biomass and lipid productivity of four oleaginous microalgae in 140-L photobioreactor outdoors. Strains
Light intensity
Lipid content (%, w/w)
Biomass productivity (mg L1 d1)
Lipid productivity (mg L1 d1)
Chlorella sp. L1
HFI MFI LFI
25.51 ± 2.96 22.17 ± 2.11 20.83 ± 1.68
137.13 ± 5.56 117.94 ± 1.39 94.97 ± 0.49
35.06 ± 5.48 26.13 ± 2.18 19.79 ± 1.70
M. dybowskii Y1
HFI MFI LFI
32.13 ± 0.08 30.52 ± 1.07 27.95 ± 1.19
86.91 ± 3.65 72.20 ± 0.92 60.49 ± 0.62
27.92 ± 1.24 22.04 ± 1.05 16.90 ± 0.54
M. dybowskii Y2
HFI MFI LFI
31.47 ± 2.68 28.56 ± 4.34 27.52 ± 4.64
106.61 ± 1.19 85.62 ± 11.87 75.97 ± 12.46
32.45 ± 4.00 23.26 ± 1.66 19.77 ± 1.29
M. dybowskii D
HFI MFI LFI
29.48 ± 0.91 28.85 ± 2.02 27.26 ± 3.40
95.67 ± 3.95 76.81 ± 3.95 60.75 ± 4.94
28.20 ± 0.30 22.16 ± 0.41 16.56 ± 0.7
crystalline compound helps fight pollution (e.g., protozoa and other microorganisms) and is often used in outdoor cultivation (Zhou et al., 2013; Xia et al., 2013; Yang et al., 2014). The results of this research clearly indicated that the four oleaginous microalgae achieved the maximum biomass productivities under 0.25 g/L urea (Fig. 2). The productivities are as follows: 162.68, 102.60, 129.66, and 83.91 mg L1 d1 in Chlorella sp. L1, M. dybowskii Y1, M. dybowskii Y2, and M. dybowskii D, respectively. Fig. 2A and C demonstrate that the biomass productivities of Chlorella sp. L1 and M. dybowskii Y2 increased with increasing urea concentrations from 0 g/L to 0.25 g/L, but decreased at 0.5 g/L. However, the biomass productivity did not decline at 0.5 g/L urea concentration in
M. dybowskii Y1 and M. dybowskii Y2 (Fig. 2B and D). These observations suggest that high nitrogen concentration may be inhibitive for the growth of microalgae (Zhou et al., 2013), which also applies to urea. Nevertheless, the tolerances of different strains to urea concentrations vary. Fig. 2 also demonstrates the changes of lipid content and productivity. The results clearly suggest that the four oleaginous microalgae achieved the highest lipid content under nitrogen-starved conditions (0 g/L urea). These findings are consistent with previous reports (Hu et al., 2008). However, no significant difference was observed in other three urea concentrations. The highest lipid productivity in Chlorella sp. L1 (42.57 mg L1 d1), M. dybowskii Y1
84
Q. He et al. / Bioresource Technology 180 (2015) 79–87
Fig. 5. Comparison the content of three lipid classes extracted from different fluctuating light intensities (HFI, MFI, LFI) by using solid phase extraction of four oleaginous microalgae: (A) Chlorella sp. L1; (B) M. dybowskii Y1; (C) M. dybowskii Y2; (D) M. dybowskii D. Different letter means the difference is significant at 0.05 level (p < 0.05).
(23.95 mg L1 d1), M. dybowskii Y2 (39.88 mg L1 d1), and M. dybowskii D (30.97 mg L1 d1) were all obtained at 0.25 g/L urea because of biomass yield. Consequently, based on lipid productivity which combined biomass productivity and lipid content, 0.25 g/L urea was considered an optimal cultivation strategy that possessed the highest yield of lipid for culture experiments outdoors.
3.4. Growth and lipid accumulation under different fluctuating light intensities outdoors The outdoor experiments were conducted in two scales of bioreactors (5-L flasks and 140-L photobioreactors) in clear summer from June to August 2013. After a 12 day culture in 5-L flasks, the highest biomass of Chlorella sp. L1, M. dybowskii Y1, M. dybowskii Y2, and M. dybowskii D were 2.54, 1.23, 2.10, and 1.26 g/L under HFI, but decreased by 44.88%, 34.15%, 46.19%, and 30.95% under LFI, respectively (Fig. 3). Similar results were identified in 140-L scales, in which all maximum biomass were obtained under HFI and were higher than those under MFI and LFI (p < 0.05) (Fig. 4). An increased biomass that corresponded to the elevated light intensity was also noted in the studies on Parietochloris incise (Solovchenko et al., 2008), Scenedesmus sp. (Liu et al., 2012), and Neochloris oleoabundans (Klok et al., 2013). The effects of different fluctuating light intensities on microalgal lipid content were analyzed. The maximum lipid content was obtained under HFI in four oleaginous microalgae in 5-L flasks (Fig. 3) and 140-L photobioreactors (Table 2). In summary, the increase of natural fluctuating light intensities resulted in enhanced biomass and lipid content in two scales of bioreactors.
The culture productivities of four oleaginous microalgae at the scales of 140-L photobioreactor were analyzed in Table 2. Chlorella sp. L1 obtained the highest biomass productivity of 137.13 mg L1 d1, followed by M. dybowskii Y2 with 106.61 mg L1 d1, whereas M. dybowskii Y1 and M. dybowskii D obtained a lower productivity around 90 mg L1 d1 under HFI. All these values were reduced by about 29% to 37% under LFI. Chlorella sp. L1 exhibited a high lipid productivity under HFI (35.06 mg L1 d1), which was 1.77 times than that under LFI (19.79 mg L1 d1). Three oleaginous microalgae of M. dybowskii also induced significant difference in lipid productivity between HFI and LFI. The comparison among four oleaginous microalgae revealed that the largest lipid productivities (35.06 and 32.45 mg L1 d1) obtained by Chlorella sp. L1 and M. dybowskii Y2 under HFI were also higher than those obtained by the other two strains under the same level of fluctuating light intensities. Therefore, these two oleaginous microalgae from biological soil crusts in desert have great potential in scale-up cultivation. The effect of light on the growth and lipid content of microalgae not only depends on strain adaptability, but also on the regime of light, particularly fluctuating light intensity. Light is the driving force in the assimilation metabolism of microalgae. Within an appropriate intensity range, light greatly promotes microalgal photosynthesis and productivity. The relationships between light and microalgae in general are as follows. (1) Under limited light intensity, the cells mainly allocate the energy on photosynthetic complexes and chloroplast membrane matrix, increasing biomass and cell membrane formation (Khotimchenko and Yakovleva, 2005; Hu et al., 2008). (2) More energy is provided with increasing light intensity to conduct the synthesis of lipid, carbohydrate, and
85
Q. He et al. / Bioresource Technology 180 (2015) 79–87
accumulation of lipid and/or TAG was proportional to light intensity up to a certain threshold, above which it did not increase because of cell damage or death caused by photoinhibition. Therefore, the scale-up cultivation cost can be reduced, and the maximum lipid productivity can be achieved by realizing how local light resources can be sufficiently used. 3.5. Lipid classes and FA composition outdoors Total lipid in microalgae is mainly composed of neutral lipids (NL), glycolipids (GL) and phospholipids (PL). The lipid classes of four oleaginous strains in 140-L photobioreactors were analyzed in Fig. 5. The cellular contents of NL, GL, and PL increased, decreased, and remained stable, respectively, in response to the elevated natural fluctuating light intensities. For example, the NL content in total lipid (37.89%) was the highest under HFI in Chlorella sp. L1. This measurement was 1.8 times than that under LFI (p < 0.05), whereas the GL content under HFI obviously reduced (p < 0.05), and PL was relatively constant (Fig. 5A). The decline of GL may be attributed to the increase of NL by remodeling the membrane lipid. By calculating the relative increment of lipid classes in dry weight (%), the increase of NL under HFI was due to the conversion of GL, accounting for 12.52%, 17.15%, 19.92%, and 11.14% in Chlorella sp. L1, M. dybowskii Y1, M. dybowskii Y2, and M. dybowskii D, respectively, without PL conversion. The remaining 80% increase was from other ways. These results conform to those obtained by existing studies on Haematococcus pluvialis (Gwak et al., 2014). Accordingly, the current research detected that the HFI not only promoted lipid content, but also improved the classes of lipid with enhanced NL content under natural light outdoors. As we know, NL is mainly composed of TAGs. GL and PL which are the main components of membrane lipids can be also convert into TAG (Pancha et al., 2014). The synthesis of NL in microalgae largely depends on nutrition or environmental stress. In the research of Burrows et al. (2012), approximately 60% of TAG was
Table 3 Fatty acid profile (%) of biodiesel in four oleaginous microalgae in 140-L photobioreactor outdoors.
Fig. 6. Variations of irradiance and air temperature of all microalgae in 5-L flasks (A, 12 days) and 140-L photobioreactor (B, 7 days). The data were obtained from a detection system which monitored the irradiance and temperature every 10 min (the average value of each day from 6:00 am to 18:00 pm during the experimental period).
protein (Liu et al., 2012). Several studies have noted that intracellular starch is disassembled to monosaccharide, such as glucose, and more lipids can be induced under high light intensities (Siaut et al., 2011; Ho et al., 2012). (3) When the light is exceedingly intense, the excited electrons at PSII exceed the delivery capacity of the photosynthetic electron transport chain, thereby markedly increasing the reactive oxygen species (ROS). This phenomenon results in the following: cells are subjected to serious oxidative stress, microalgal growth is arrested, photosynthetic activity decreases, and excess energy is stored in the form of lipid and/or TAG (Zhang et al., 2013) because lipid can use more energy than other compounds (e.g., starch). Lipid also has a certain degree of reflecting and shielding functions to cope with high light. Given these findings, the increase of lipid is considered an adaption mechanism of microalgae to high light intensity. However, the results of the same lipid and/or TAG content in different light intensities observed by Breuer et al. (2013) may be attributed to the limited setting regime of light intensities. Nonetheless, the
Fatty acids
Chlorella sp. L1
M. dybowskii Y1
M. dybowskii Y2
C C C C C C C
1.4 0.57 30.29 5.97 4.03 32.41 13.2 0.46 10.09 0.33 ND 0.32 0.93 96.78
0.86 0.32 38.06 2.05 5.33 31.73 6.89 4.19 7.37 1.09 0.13 0.84 1.14 96.71
1.11 0.54 51.85 8.91 1.14 23.49 1.55 6.06 2.51 0.73 ND 0.1 2.01 96.24
1.45 0.76 33.53 3.91 1.53 21.29 11.32 1.29 17.75 6.28 0.09 0.07 0.73 96. 90
36.05 38.95 24.08
45.34 34.1 19.54
54.83 32.94 10.85
42.79 25.96 36.64
0.97 4.59
0.79 4.71
0.66 4.79
1.43 4.30
56.41 84.83 34.75 15.39
57.64 71.15 33.81 15.41
58.45 62.02 25.70 15.67
14:0 14:1 16:0 16:1 18:0 18:1 18:2 c C-18:3 a C-18:3 C 18:4 C 20:0 C 22:0 Others C16-C18 (% total FAME) P SFA P MUFA P PUFA Biodiesel properties DU Viscosity 40 °C (mm2 s1) CN IV (g I2 100 g1) LCSF (wt.%) CFPP (°C)
M. dybowskii D
53.32 119.24 33.80 15.42
ND: not detected; DU: degree of unsaturation; CN: cetane number; IV: Iodine value; LCSF: long-chain saturated factor; CFPP: cold filter plugging point.
86
Q. He et al. / Bioresource Technology 180 (2015) 79–87
Table 4 Comparisons of biomass and lipid productivity of different scales in some microalgae outdoors. Strains
LC (%, w/w)
VBP (mg L1 d1)
ABP (g m2 d1)
VLP (mg L1 d1)
ALP (g m2 d1)
Bioreactor
References
C. zofingiensis Chlorella sp. NJ-18 Nannochloropsis sp. S. obtusus XJ-15 M. dybowskii LB50 Nannochloropsis sp. Nannochloropsis sp. Nannochloropsis sp. Chlorella sp. L1 M. dybowskii Y1 M. dybowskii Y2 M. dybowskii D
54.50 27.89 60 42.10 38.57 16–25 35 18% 25.51 32.13 31.47 29.48
40.90 91.84 300 86.50 81.43 23 – – 137.13 86.91 106.61 95.67
– – – 21.40 21.71 3.47 7.40 10.00 35.52 22.49 28.43 25.51
22.30 25.04 204.00 23.20 32.48 6 – – 35.06 27.92 33.55 28.20
– – – – 8.59 – – – 9.06 7.22 8.95 7.52
60 L FPP 70 L PBR 110 L PBR 140 L PBR 140 L PBR 7500 L ORP PBR PBR 140 L PBR 140 L PBR 140 L PBR 140 L PBR
Feng et al. (2011) Zhou et al. (2013) Rodolfi et al. (2009) Xia et al. (2013) Yang et al. (2014) Richardson et al. (2014) Richardson et al. (2014) Richardson et al. (2014) This study This study This study This study
LC: lipid content; VBP: volumetric biomass productivity; ABP: areal biomass productivity; VLP: volumetric lipid productivity; ALP: areal lipid productivity; FPP: flat plate photobioreactors; PBR: photobioreactor; ORP: open race way pond.
synthesized from de novo carbon fixation under nitrate deprivation stress. Meanwhile, the remaining 40% of TAG was obtained from the transformation of pigment, protein, starch, and other components of lipid membranes (Pal et al., 2011; Burrows et al., 2012; Ho et al., 2012; Ge et al., 2014; Li et al., 2014; Pancha et al., 2014; Takeshita et al., 2014). Under the induction of high alkalinity in pH (Xia et al., 2014) or NaCl (Yang et al., 2014), both TAG and PL all increased, in which the majority of the TAG was generated because of de novo fatty acid biosynthesis, and 10% to 20% of TAG was induced by GL transformation. In terms of light intensity, the up-regulation of de novo fatty acid biosynthesis was realized at the gene expression level under high light intensity (Gwak et al., 2014). At the same time, the growth and photoprotection ability of microalgae increased (Klok et al., 2013). Therefore, we speculate that when the growth of microalgae was not evidently inhibited under HFI outdoors, a large proportion of TAG was accumulated via de novo fatty acid biosynthesis, and about 11% to 20% of TAG was formed via GL transformation. Meanwhile, the studies that performed indoor experiments with constant high light intensity determined that PL was reduced and was converted into NL (Pancha et al., 2014). This finding was not identified in the outdoor fluctuating intensities in the current study. Such phenomenon may be a major reason for the decline of lipid content, which was cultivated by previous studies in scale-up outdoor experiments. The FA composition and important properties of biodiesel (summarized in Table 3), which can be potentially obtained from four microalgae growing in HFI of 140-L scales, were estimated to further examine the quality of biodiesel. The most abundant synthesized FAMEs were FAs with C16–C18 (>95%) in four microalgae. These FAs are key components for producing biodiesel (Zhou et al., 2013; Xia et al., 2013., Olmstead et al., 2013; Takeshita et al., 2014). Monounsaturated fatty acids (i.e., C18:1) have a large proportion, essentially consisting of NLs, and favor biodiesel production (Hu et al., 2008). The relevant biodiesel properties, such as the degree of unsaturation, viscosity, iodine value (IV), cetane number (CN), and cold filter plugging point (CFPP), were determined with FAME profile. The biodiesel of four microalgae had a viscosity value lower than the maximum value (
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Sufficient utilization of natural fluctuating light intensity is an effective approach of promoting lipid productivity in oleaginous microalgal cultivation outdoors Qiaoning He a,b, Haijian Yang a,b, Liangliang Xu a,b, Ling Xia a, Chunxiang Hu a,⇑ a b
Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China University of Chinese Academy of Sciences, Beijing 100049, China
h i g h l i g h t s Six microalgae were identified and analyzed for growth and lipid accumulation. 0.25 g L
1
urea was optimal concentration for culture outdoors.
Lipid accumulation under natural fluctuating light intensities was investigated. Lipid yield and neutral lipid content were enhanced by high fluctuating light intensity. The reduction in glycolipids contributed to NL accumulation under HFI.
a r t i c l e
i n f o
Article history: Received 31 October 2014 Received in revised form 23 December 2014 Accepted 24 December 2014 Available online 31 December 2014 Keywords: Microalgae Fluctuating light intensity Lipid productivity Neutral lipid Glycolipid
a b s t r a c t The effects of fluctuating intensity of solar radiation on biomass and lipid in oleaginous microalgae are important. However, this topic has not been the subject of studies for a long time. In this study, four oleaginous microalgae from semi-arid areas were screened and cultivated outdoors under different fluctuating intensities. Results showed that the highest lipid productivities and neutral lipid (NL) contents occurred under high fluctuating intensity (HFI), in which 13–20% of the increased NL came from glycolipid transformation without phospholipid conversion. Chlorella sp. L1 and Monoraphidium dybowskii Y2 obtained from biological soil crusts in desert had the largest biomass (137.13, 106.61 mg L1 d1) and lipid yields (35.06, 32.45 mg L1 d1) under HFI. The highest areal lipid productivities of 9.06 and 8.95 g m2 d1 and better biodiesel quality were observed under HFI. Accordingly, sufficiently adopting fluctuating light intensity outdoors to culture microalgae was an economic and effective approach. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Given the rapid depletion of fossil fuels and serious environmental problems caused by the extensive use of fossil fuels resources, exploring and employing alternative and environmentally friendly energy resources are receiving considerable attention (William and Laurens, 2010). Alternative energy resources have also become an important strategic direction of global energy structure. Microalgae with various advantages are currently regarded as one of the effective and exceedingly optimistic alternative energy resources (Hu et al., 2008; Griffiths and Harrison, 2009; Siaut et al., 2011). However, cultivating microalgae at large-scale remains to be a problem (Rodolfi et al., 2009), particularly the lipid ⇑ Corresponding author. Tel./fax: +86 27 68780866. E-mail address: [email protected] (C. Hu). http://dx.doi.org/10.1016/j.biortech.2014.12.088 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
content decreases with increasing cultivation (Yang et al., 2014). Lipid is biologically considered an energy storage of microalgae; its production generally increases as a result of exposure to stressful environment, such as nitrogen, phosphorus, and iron deficiency, high salinity, and high pH etc. (Courchesne et al., 2009). Although the lipid content is enhanced, biomass is usually low and lipid productivity is constrained under such condition. Light is the fundamental driving force in the growth of photoautrophic microalgae. In particular, light significantly affects microalgal growth and lipid accumulation (Solovchenko et al., 2008; Liu et al., 2012). Among the effects of light, fluctuations in light intensity are one of the primary factors that influence the generation of high biomass and lipid content in microalgae (Wahidin et al., 2013). Therefore, the sufficient and effective utilization of sunlight during outdoor cultivation deemed extremely potential. However, previous studies presented conflicting results. Some observations
80
Q. He et al. / Bioresource Technology 180 (2015) 79–87
noted that low light intensity results in the accumulation of lipids and/or triacylglycerols (TAG) (Breuer et al., 2013). By contrast, other studies specified that lipid content can be enhanced by high light intensity (Li et al., 2012; Liu et al., 2012), particularly neutral lipids (NLs) (Sharma et al., 2012; Gwak et al., 2014), and low light essentially increases polar lipids (Hu et al., 2008). Outdoor cultures are subjected daily to cyclic changes in dramatic fluctuations of light intensity. In addition to weather conditions and diurnal cycle, the capability of harvesting light and self-shading in microalgae and the reactor translucent influence lipid accumulation in mass cultivation (Pulz et al., 2001; Simionato et al., 2013). Culture productivity is invariably controlled by the availability of light, particularly when the scale of photobioreactor increases. In fact, more people realize the challenge between cost and productivity in microalgae biodiesel. Thus, according to the local natural light resource, maximizing areal lipid productivity should be a parameter that is better associated for industrializing microalgae biofuels. Investigating the influence of natural light fluctuating intensity on the cultivation of oleaginous microalgae is rarely tested outdoors with photobioreactors; thus, it is still exceedingly relevant to be thoroughly explored. Given the above conditions, this research intended to realize the following objectives: (1) identify the strains of microalgae isolated from the topsoil of semi-arid areas through morphological features and 18S rDNA sequences; (2) examine their potential in oilproducing; (3) optimize nitrogen concentrations in the form of low-cost urea outdoors; and (4) determine the effect of natural fluctuating light intensity on growth, lipid content, and lipid classes at both 5 L and 140 L scales to obtain the highest lipid productivity with better biodiesel quality in unit area by reasonable arrangement of photobioreactors. 2. Methods 2.1. Strains The six microalgae used in this study were isolated from the topsoil of semi-arid areas in the northwest of China. Among them, Chlorella sp. L1 and Monoraphidium dybowskii Y2 were obtained from the biological soil crusts of Tengger desert in Ningxia (37°280 N, 105°000 E); M. dybowskii Y1, M. dybowskii D, Chlorella sp. X, and Nannochloris sp. L2 were obtained from the Loess Plateau in Lanzhou City of Gansu (36°020 N, 103°490 E). All microalgae were stored with BG11 medium in FACHB-Collection (Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China). 2.2. Experimental design 2.2.1. Identification Six strains of microalgae were initially identified by morphology under a microscope and were further determined with 18S rDNA sequences. The total genomic DNA was extracted from algal cells with a DNA isolation kit (TianGen Biotech (Bei Jing) Co., Ltd, China). Polymerase chain reaction (PCR) was performed with general primers 18S-1 (50 -tggttgatcctgccagtagtc–30 and 18S-2 (50 -tgatccttctgcaggttcacc-30 ) to amplify 18S rDNA gene. The PCR products were excised from agarose gel, recovered with the DNA isolation kit, and were then sequenced. Finally phylogenetic analyses were conducted using the NCBI Gene Bank database. 2.2.2. Preliminary screen indoors Six strains of microalgae were cultivated with 400 mL of BG-11 medium in 500 mL Erlenmeyer flasks. The initial OD was 0.3. Cool white fluorescent tubes acted as light source to provide the light
Fig. 1. Maximum-likelihood tree of six microalgae inferred from 18S rDNA gene sequence (strains obtained during this study are underlined). Bootstrap values are shown at the internal nodes for neighbor joining (5000 replications), maximum parsimony (1000 replications) and maximum likelihood (100 replications), respectively, if the node is supported by at least two bootstrap values of 50% or above. Branch lengths correspond to evolutionary distances. A distance of 2 is indicated by the scale.
intensity of 100 lmol photons m2 s1, and the temperature was maintained at 25 ± 1 °C with an air conditioner. Filtered air was supplied to flasks by using an air compressor. 2.2.3. Cultivation outdoors The candidate strains were selected based on their lipid productivity. Scale-up experiments were performed during summer in the bioreactors (5-L flask and 140-L photobioreactor), which were placed inside a green house in Beijing, China (40°220 N, 116°200 E). Aeration in 5-L flasks was at a flow rate of 4 L min/L, and the reactor was stirred with a 5 cm magnetic stir bar (mixing at 150 rpm) at the middle of the reactor. The 140-L photobioreactor was composed of two connected 70-L polyvinylchloride hanging bags (22 cm diameter, 180 cm height) (Dalian Huixin Titanium Equipment Development, Co., Ltd) (Xia et al., 2013). The spatial layout was equipped with 80 photobioreactors in an area of 42 m2. Aeration with 1.8% CO2 was provided to the photobioreactors by using an air compressor as carbon source at daytime and as pure air at night. The temperature in the greenhouse was controlled by an air conditioner at a constant range between 25 °C and 28 °C. 2.2.3.1. Optimization of urea concentration in 5-L flasks outdoors. The BG-11 medium was used outdoors, but the form of nitrogen source in the culture was substituted by urea, which was optimized in 5-L flasks outdoors. The urea concentrations were set at 0, 0.1, 0.25, and 0.5 g/L. The screened four oleaginous microalgae were cultivated with 3.5 L of modified BG11 medium in 5-L flasks at different urea concentrations to induce lipid accumulation. The microalgae were harvested when the cultures reached the late exponential phase. 2.2.3.2. Experiments on natural fluctuating light intensity. Four oleaginous microalgae were operated in 5-L flasks and 140-L photobioreactors with the optimal 0.25 g/L urea concentration. Three different natural fluctuating light intensity levels were provided to examine the effects of natural fluctuating light intensity. In
81
Q. He et al. / Bioresource Technology 180 (2015) 79–87 Table 1 Biomass and lipid production of six microalgae. Strains 1
Biomass (g L ) Biomass productivity (mg L1 d1) Lipid content (%, w/w) Lipid productivity (mg L1 d1)
M. dybowskii D
M. dybowskii Y1
M. dybowskii Y2
Chlorella sp. L1
Chlorella sp. X
Nannochloris sp. L2
1.89 ± 0.08 125.79 ± 5.46 33.09 ± 1.98 41.64 ± 3.33
1.79 ± 0.06 121.16 ± 4.87 33.07 ± 2.61 40.01 ± 2.30
2.36 ± 0.10 157.20 ± 6.79 34.63 ± 1.27 54.39 ± 0.38
2.67 ± 0.08 178.03 ± 5.55 26.92 ± 1.39 47.87 ± 0.95
2.48 ± 0.05 165.63 ± 3.01 19.01 ± 3.21 34.53 ± 0.44
2.54 ± 0.13 169.28 ± 0.90 18.52 ± 1.31 31.92 ± 2.97
Data are the means and standard deviation of three independent experiments, the same below.
Fig. 2. Effect of different urea concentrations on biomass and lipid accumulation in four oleaginous microalgae: (A) Chlorella sp. L1; (B) M. dybowskii Y1; (C) M. dybowskii Y2; (D) M. dybowskii D in 5-L flasks outdoors. BP: biomass productivity; LP: lipid productivity.
particular, these light intensity levels were divided into high fluctuating intensity (HFI), middle fluctuating intensity (MFI, about 60% of HFI) and low fluctuating intensity (LFI, about 30% of HFI). Placing the 5-L flasks on a platform of three different layers and in 140-L photobioreactor, directions, and inclinations were remarkably important in absorbing light, particularly via dislocation arrangement and shading to satisfy the requirements. Irradiance and temperature were measured with a detection system, which recorded the data every 10 min of an average value from 6:00 to 18:00 (Fig. 6). The maximum light intensity in daytime was fluctuated from 990 lmol photons m2 s1 to 1486 lmol photons m2 s1. The average light intensity of every level was changed by the weather, but the same level was uniform in the two types of bioreactors. All experiments were conducted in triplicate from June to August 2013. 2.3. Analytical procedures 2.3.1. Biomass measurement Cells density was determined by measuring optical density at 680 nm (OD680). The following below equations describe the
relationships between dry weight (DW, g/L) and the OD680 values of six strains of microalgae. DW = 0.329 OD680, DW = 0.266 OD680, DW = 0.172 OD680, DW = 0.226 OD680, DW = 0.188 OD680, DW = 0.317 OD680,
R2 = 0.947 R2 = 0.986 R2 = 0.994 R2 = 0.984 R2 = 0.989 R2 = 0.996
(Chlorella sp. L1) (Chlorella sp. X) (M. dybowskii Y1) (M. dybowskii Y2) (M. dybowskii D) (Nannochloris sp. L2)
The microalgae were then harvested by centrifugation and were lyophilized with a vacuum freeze dryer (Alpha 1-2 LD plus, Christ). 2.3.2. Lipid analysis Lipid content: Total lipid was extracted using a Soxhlet reflux extractor with chloroform/methanol (2/1, v/v) that ranged from 50 mg to 100 mg of lyophilized algae, and was then gravimetrically quantified. Lipid classes: Solid phase extraction (SPE) was utilized to separate lipid extracts into three fractions, namely, neutral lipids
82
Q. He et al. / Bioresource Technology 180 (2015) 79–87
Fig. 3. Effect of different fluctuating light intensities on growth and lipid content in four oleaginous microalgae: (A) Chlorella sp. L1; (B) M. dybowskii Y1; (C) M. dybowskii Y2; (D) M. dybowskii D in 5-L flasks outdoors.
(NL), glycolipids (GL) and phospholipids (PL). In particular, SPE was conducted with a column (20 mm 200 mm) containing 4 g of silica cartridges. The extracted lipids (approximately 100 mg) were dissolved in 400 lL of chloroform and were fractionated by using the following solvent systems: neutral lipids with chloroform (six column volumes), glycolipids with acetone: methanol (9:1, v/v) (four column volumes) and phospholipids with methanol (four column volumes). The eluted lipid fractions were then dried under nitrogen at 40 °C, and the mass of each fraction was recorded. Fatty acids (FA) composition: Biodiesel was determined as fatty acid methyl esters (FAMEs) after acidic transesterification of lipids. The fatty acid components of extracted microalgal oil were analyzed by gas chromatograph mass spectrometry (GC–MS; Thermo Scientific ITQ 700, USA) equipped with a flame ionization detector (FID) and a fused silica capillary column (60 m 0.25 mm 0.25 lm; Agilent Technologies, USA). The injector and detector temperatures were maintained at 270 °C and 280 °C, respectively, with an oven temperature gradient of 50 °C to 170 °C at 40 °C min1 after a 1 min hold time at 50 °C, then with an oven temperature gradient of 170 °C to 210 °C at 18 °C min1 after a 1 min hold for 1 min. All parameters of the FAMEs were derived from the calibration curves generated from the FAME standard mix (Supelco 37 component FAME mix, Sigma–Aldrich, USA).
2.3.3. Statistical analysis All values were expressed as the mean ± standard deviation. The data were analyzed by two-way ANOVA using the SPSS statistical software (version 19.0). p < 0.05 was considered to have a statistically significant difference.
3. Results and discussion 3.1. Identification of microalgae The six strains of microalgae isolated from the topsoil of semiarid areas were all identified as green algae by morphological characterization. The 18S rDNA sequences of these strains were then further compared. The results revealed the affiliation of strains L1 and X to Chlorella sp., strains D, Y1, and Y2 to M. dybowskii, and strain L2 to Nannochloris sp. (Fig. 1). 3.2. Strain selection indoors The findings on six microalgae cultured indoors (Table 1) demonstrated that the highest biomass productivity was 178.03 mg L1 d1 in Chlorella sp. L1, followed by Nannochloris sp. L2, Chlorella sp. X, and three M. dybowskii strains. The highest lipid content was observed in M. dybowskii Y2 at 34.63%. The best lipid producer was the strain that possessed the best combination of biomass productivity and lipid content; thus, lipid productivity was the most reliable indicator for oleaginous microalgae. (Rodolfi et al., 2009; Griffiths and Harrison, 2009). M. dybowskii Y2 with maximum lipid productivity (54.39 mg L1 d1), followed by Chlorella sp. L1 (47.87 mg L1 d1), M. dybowskii D (41.64 mg L1 d1), and M. dybowskii Y1 (40.01 mg L1 d1), which all had lipid productivity of more than 40 mg L1 d1, were selected as the potential feedstock for the subsequent scale-up experiments. 3.3. Optimization of urea concentrations outdoors Urea is considered a good choice for large-scale production because of its low cost (Matsudo et al., 2009). Moreover, this
83
Q. He et al. / Bioresource Technology 180 (2015) 79–87
Fig. 4. Effect of different fluctuating light intensities on growth in four oleaginous microalgae: (A) Chlorella sp. L1; (B) M. dybowskii Y1; (C) M. dybowskii Y2; (D) M. dybowskii D in 140-L photobioreactor outdoors.
Table 2 Biomass and lipid productivity of four oleaginous microalgae in 140-L photobioreactor outdoors. Strains
Light intensity
Lipid content (%, w/w)
Biomass productivity (mg L1 d1)
Lipid productivity (mg L1 d1)
Chlorella sp. L1
HFI MFI LFI
25.51 ± 2.96 22.17 ± 2.11 20.83 ± 1.68
137.13 ± 5.56 117.94 ± 1.39 94.97 ± 0.49
35.06 ± 5.48 26.13 ± 2.18 19.79 ± 1.70
M. dybowskii Y1
HFI MFI LFI
32.13 ± 0.08 30.52 ± 1.07 27.95 ± 1.19
86.91 ± 3.65 72.20 ± 0.92 60.49 ± 0.62
27.92 ± 1.24 22.04 ± 1.05 16.90 ± 0.54
M. dybowskii Y2
HFI MFI LFI
31.47 ± 2.68 28.56 ± 4.34 27.52 ± 4.64
106.61 ± 1.19 85.62 ± 11.87 75.97 ± 12.46
32.45 ± 4.00 23.26 ± 1.66 19.77 ± 1.29
M. dybowskii D
HFI MFI LFI
29.48 ± 0.91 28.85 ± 2.02 27.26 ± 3.40
95.67 ± 3.95 76.81 ± 3.95 60.75 ± 4.94
28.20 ± 0.30 22.16 ± 0.41 16.56 ± 0.7
crystalline compound helps fight pollution (e.g., protozoa and other microorganisms) and is often used in outdoor cultivation (Zhou et al., 2013; Xia et al., 2013; Yang et al., 2014). The results of this research clearly indicated that the four oleaginous microalgae achieved the maximum biomass productivities under 0.25 g/L urea (Fig. 2). The productivities are as follows: 162.68, 102.60, 129.66, and 83.91 mg L1 d1 in Chlorella sp. L1, M. dybowskii Y1, M. dybowskii Y2, and M. dybowskii D, respectively. Fig. 2A and C demonstrate that the biomass productivities of Chlorella sp. L1 and M. dybowskii Y2 increased with increasing urea concentrations from 0 g/L to 0.25 g/L, but decreased at 0.5 g/L. However, the biomass productivity did not decline at 0.5 g/L urea concentration in
M. dybowskii Y1 and M. dybowskii Y2 (Fig. 2B and D). These observations suggest that high nitrogen concentration may be inhibitive for the growth of microalgae (Zhou et al., 2013), which also applies to urea. Nevertheless, the tolerances of different strains to urea concentrations vary. Fig. 2 also demonstrates the changes of lipid content and productivity. The results clearly suggest that the four oleaginous microalgae achieved the highest lipid content under nitrogen-starved conditions (0 g/L urea). These findings are consistent with previous reports (Hu et al., 2008). However, no significant difference was observed in other three urea concentrations. The highest lipid productivity in Chlorella sp. L1 (42.57 mg L1 d1), M. dybowskii Y1
84
Q. He et al. / Bioresource Technology 180 (2015) 79–87
Fig. 5. Comparison the content of three lipid classes extracted from different fluctuating light intensities (HFI, MFI, LFI) by using solid phase extraction of four oleaginous microalgae: (A) Chlorella sp. L1; (B) M. dybowskii Y1; (C) M. dybowskii Y2; (D) M. dybowskii D. Different letter means the difference is significant at 0.05 level (p < 0.05).
(23.95 mg L1 d1), M. dybowskii Y2 (39.88 mg L1 d1), and M. dybowskii D (30.97 mg L1 d1) were all obtained at 0.25 g/L urea because of biomass yield. Consequently, based on lipid productivity which combined biomass productivity and lipid content, 0.25 g/L urea was considered an optimal cultivation strategy that possessed the highest yield of lipid for culture experiments outdoors.
3.4. Growth and lipid accumulation under different fluctuating light intensities outdoors The outdoor experiments were conducted in two scales of bioreactors (5-L flasks and 140-L photobioreactors) in clear summer from June to August 2013. After a 12 day culture in 5-L flasks, the highest biomass of Chlorella sp. L1, M. dybowskii Y1, M. dybowskii Y2, and M. dybowskii D were 2.54, 1.23, 2.10, and 1.26 g/L under HFI, but decreased by 44.88%, 34.15%, 46.19%, and 30.95% under LFI, respectively (Fig. 3). Similar results were identified in 140-L scales, in which all maximum biomass were obtained under HFI and were higher than those under MFI and LFI (p < 0.05) (Fig. 4). An increased biomass that corresponded to the elevated light intensity was also noted in the studies on Parietochloris incise (Solovchenko et al., 2008), Scenedesmus sp. (Liu et al., 2012), and Neochloris oleoabundans (Klok et al., 2013). The effects of different fluctuating light intensities on microalgal lipid content were analyzed. The maximum lipid content was obtained under HFI in four oleaginous microalgae in 5-L flasks (Fig. 3) and 140-L photobioreactors (Table 2). In summary, the increase of natural fluctuating light intensities resulted in enhanced biomass and lipid content in two scales of bioreactors.
The culture productivities of four oleaginous microalgae at the scales of 140-L photobioreactor were analyzed in Table 2. Chlorella sp. L1 obtained the highest biomass productivity of 137.13 mg L1 d1, followed by M. dybowskii Y2 with 106.61 mg L1 d1, whereas M. dybowskii Y1 and M. dybowskii D obtained a lower productivity around 90 mg L1 d1 under HFI. All these values were reduced by about 29% to 37% under LFI. Chlorella sp. L1 exhibited a high lipid productivity under HFI (35.06 mg L1 d1), which was 1.77 times than that under LFI (19.79 mg L1 d1). Three oleaginous microalgae of M. dybowskii also induced significant difference in lipid productivity between HFI and LFI. The comparison among four oleaginous microalgae revealed that the largest lipid productivities (35.06 and 32.45 mg L1 d1) obtained by Chlorella sp. L1 and M. dybowskii Y2 under HFI were also higher than those obtained by the other two strains under the same level of fluctuating light intensities. Therefore, these two oleaginous microalgae from biological soil crusts in desert have great potential in scale-up cultivation. The effect of light on the growth and lipid content of microalgae not only depends on strain adaptability, but also on the regime of light, particularly fluctuating light intensity. Light is the driving force in the assimilation metabolism of microalgae. Within an appropriate intensity range, light greatly promotes microalgal photosynthesis and productivity. The relationships between light and microalgae in general are as follows. (1) Under limited light intensity, the cells mainly allocate the energy on photosynthetic complexes and chloroplast membrane matrix, increasing biomass and cell membrane formation (Khotimchenko and Yakovleva, 2005; Hu et al., 2008). (2) More energy is provided with increasing light intensity to conduct the synthesis of lipid, carbohydrate, and
85
Q. He et al. / Bioresource Technology 180 (2015) 79–87
accumulation of lipid and/or TAG was proportional to light intensity up to a certain threshold, above which it did not increase because of cell damage or death caused by photoinhibition. Therefore, the scale-up cultivation cost can be reduced, and the maximum lipid productivity can be achieved by realizing how local light resources can be sufficiently used. 3.5. Lipid classes and FA composition outdoors Total lipid in microalgae is mainly composed of neutral lipids (NL), glycolipids (GL) and phospholipids (PL). The lipid classes of four oleaginous strains in 140-L photobioreactors were analyzed in Fig. 5. The cellular contents of NL, GL, and PL increased, decreased, and remained stable, respectively, in response to the elevated natural fluctuating light intensities. For example, the NL content in total lipid (37.89%) was the highest under HFI in Chlorella sp. L1. This measurement was 1.8 times than that under LFI (p < 0.05), whereas the GL content under HFI obviously reduced (p < 0.05), and PL was relatively constant (Fig. 5A). The decline of GL may be attributed to the increase of NL by remodeling the membrane lipid. By calculating the relative increment of lipid classes in dry weight (%), the increase of NL under HFI was due to the conversion of GL, accounting for 12.52%, 17.15%, 19.92%, and 11.14% in Chlorella sp. L1, M. dybowskii Y1, M. dybowskii Y2, and M. dybowskii D, respectively, without PL conversion. The remaining 80% increase was from other ways. These results conform to those obtained by existing studies on Haematococcus pluvialis (Gwak et al., 2014). Accordingly, the current research detected that the HFI not only promoted lipid content, but also improved the classes of lipid with enhanced NL content under natural light outdoors. As we know, NL is mainly composed of TAGs. GL and PL which are the main components of membrane lipids can be also convert into TAG (Pancha et al., 2014). The synthesis of NL in microalgae largely depends on nutrition or environmental stress. In the research of Burrows et al. (2012), approximately 60% of TAG was
Table 3 Fatty acid profile (%) of biodiesel in four oleaginous microalgae in 140-L photobioreactor outdoors.
Fig. 6. Variations of irradiance and air temperature of all microalgae in 5-L flasks (A, 12 days) and 140-L photobioreactor (B, 7 days). The data were obtained from a detection system which monitored the irradiance and temperature every 10 min (the average value of each day from 6:00 am to 18:00 pm during the experimental period).
protein (Liu et al., 2012). Several studies have noted that intracellular starch is disassembled to monosaccharide, such as glucose, and more lipids can be induced under high light intensities (Siaut et al., 2011; Ho et al., 2012). (3) When the light is exceedingly intense, the excited electrons at PSII exceed the delivery capacity of the photosynthetic electron transport chain, thereby markedly increasing the reactive oxygen species (ROS). This phenomenon results in the following: cells are subjected to serious oxidative stress, microalgal growth is arrested, photosynthetic activity decreases, and excess energy is stored in the form of lipid and/or TAG (Zhang et al., 2013) because lipid can use more energy than other compounds (e.g., starch). Lipid also has a certain degree of reflecting and shielding functions to cope with high light. Given these findings, the increase of lipid is considered an adaption mechanism of microalgae to high light intensity. However, the results of the same lipid and/or TAG content in different light intensities observed by Breuer et al. (2013) may be attributed to the limited setting regime of light intensities. Nonetheless, the
Fatty acids
Chlorella sp. L1
M. dybowskii Y1
M. dybowskii Y2
C C C C C C C
1.4 0.57 30.29 5.97 4.03 32.41 13.2 0.46 10.09 0.33 ND 0.32 0.93 96.78
0.86 0.32 38.06 2.05 5.33 31.73 6.89 4.19 7.37 1.09 0.13 0.84 1.14 96.71
1.11 0.54 51.85 8.91 1.14 23.49 1.55 6.06 2.51 0.73 ND 0.1 2.01 96.24
1.45 0.76 33.53 3.91 1.53 21.29 11.32 1.29 17.75 6.28 0.09 0.07 0.73 96. 90
36.05 38.95 24.08
45.34 34.1 19.54
54.83 32.94 10.85
42.79 25.96 36.64
0.97 4.59
0.79 4.71
0.66 4.79
1.43 4.30
56.41 84.83 34.75 15.39
57.64 71.15 33.81 15.41
58.45 62.02 25.70 15.67
14:0 14:1 16:0 16:1 18:0 18:1 18:2 c C-18:3 a C-18:3 C 18:4 C 20:0 C 22:0 Others C16-C18 (% total FAME) P SFA P MUFA P PUFA Biodiesel properties DU Viscosity 40 °C (mm2 s1) CN IV (g I2 100 g1) LCSF (wt.%) CFPP (°C)
M. dybowskii D
53.32 119.24 33.80 15.42
ND: not detected; DU: degree of unsaturation; CN: cetane number; IV: Iodine value; LCSF: long-chain saturated factor; CFPP: cold filter plugging point.
86
Q. He et al. / Bioresource Technology 180 (2015) 79–87
Table 4 Comparisons of biomass and lipid productivity of different scales in some microalgae outdoors. Strains
LC (%, w/w)
VBP (mg L1 d1)
ABP (g m2 d1)
VLP (mg L1 d1)
ALP (g m2 d1)
Bioreactor
References
C. zofingiensis Chlorella sp. NJ-18 Nannochloropsis sp. S. obtusus XJ-15 M. dybowskii LB50 Nannochloropsis sp. Nannochloropsis sp. Nannochloropsis sp. Chlorella sp. L1 M. dybowskii Y1 M. dybowskii Y2 M. dybowskii D
54.50 27.89 60 42.10 38.57 16–25 35 18% 25.51 32.13 31.47 29.48
40.90 91.84 300 86.50 81.43 23 – – 137.13 86.91 106.61 95.67
– – – 21.40 21.71 3.47 7.40 10.00 35.52 22.49 28.43 25.51
22.30 25.04 204.00 23.20 32.48 6 – – 35.06 27.92 33.55 28.20
– – – – 8.59 – – – 9.06 7.22 8.95 7.52
60 L FPP 70 L PBR 110 L PBR 140 L PBR 140 L PBR 7500 L ORP PBR PBR 140 L PBR 140 L PBR 140 L PBR 140 L PBR
Feng et al. (2011) Zhou et al. (2013) Rodolfi et al. (2009) Xia et al. (2013) Yang et al. (2014) Richardson et al. (2014) Richardson et al. (2014) Richardson et al. (2014) This study This study This study This study
LC: lipid content; VBP: volumetric biomass productivity; ABP: areal biomass productivity; VLP: volumetric lipid productivity; ALP: areal lipid productivity; FPP: flat plate photobioreactors; PBR: photobioreactor; ORP: open race way pond.
synthesized from de novo carbon fixation under nitrate deprivation stress. Meanwhile, the remaining 40% of TAG was obtained from the transformation of pigment, protein, starch, and other components of lipid membranes (Pal et al., 2011; Burrows et al., 2012; Ho et al., 2012; Ge et al., 2014; Li et al., 2014; Pancha et al., 2014; Takeshita et al., 2014). Under the induction of high alkalinity in pH (Xia et al., 2014) or NaCl (Yang et al., 2014), both TAG and PL all increased, in which the majority of the TAG was generated because of de novo fatty acid biosynthesis, and 10% to 20% of TAG was induced by GL transformation. In terms of light intensity, the up-regulation of de novo fatty acid biosynthesis was realized at the gene expression level under high light intensity (Gwak et al., 2014). At the same time, the growth and photoprotection ability of microalgae increased (Klok et al., 2013). Therefore, we speculate that when the growth of microalgae was not evidently inhibited under HFI outdoors, a large proportion of TAG was accumulated via de novo fatty acid biosynthesis, and about 11% to 20% of TAG was formed via GL transformation. Meanwhile, the studies that performed indoor experiments with constant high light intensity determined that PL was reduced and was converted into NL (Pancha et al., 2014). This finding was not identified in the outdoor fluctuating intensities in the current study. Such phenomenon may be a major reason for the decline of lipid content, which was cultivated by previous studies in scale-up outdoor experiments. The FA composition and important properties of biodiesel (summarized in Table 3), which can be potentially obtained from four microalgae growing in HFI of 140-L scales, were estimated to further examine the quality of biodiesel. The most abundant synthesized FAMEs were FAs with C16–C18 (>95%) in four microalgae. These FAs are key components for producing biodiesel (Zhou et al., 2013; Xia et al., 2013., Olmstead et al., 2013; Takeshita et al., 2014). Monounsaturated fatty acids (i.e., C18:1) have a large proportion, essentially consisting of NLs, and favor biodiesel production (Hu et al., 2008). The relevant biodiesel properties, such as the degree of unsaturation, viscosity, iodine value (IV), cetane number (CN), and cold filter plugging point (CFPP), were determined with FAME profile. The biodiesel of four microalgae had a viscosity value lower than the maximum value (
