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Enhancing lipid productivity by co-cultivation of Chlorella sp. U4341 and Monoraphidium sp. FXY-10
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e6, 2014 www.elsevier.com/locate/jbiosc
Enhancing lipid productivity by co-cultivation of Chlorella sp. U4341 and Monoraphidium sp. FXY-10 Peng Zhao,1, 2 Xuya Yu,1, 2 Junjun Li,1 Xianhua Tang,1 and Zunxi Huang1, 3, * Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Kunming 650500, China,1 College of Life Sciences and Biotechnology, Kunming University of Science and Technology, Kunming 650500, China,2 and School of Life Sciences, Yunnan Normal University, Kunming 650500, China3 Received 9 September 2013; accepted 18 December 2013 Available online xxx
To improve lipid productivity, co-cultivation of Chlorella sp. U4341 and Monoraphidium sp. FXY-10 for lipid production was studied. Compared with mono-cultivations, co-cultivation of the two microalgae significantly increased the accumulation of total biomass and total lipid yield, and enhanced the lipid productivity (29.52 mg LL1 dL1). Fatty acid compositions significantly varied in different cultivations. The content of C18 fatty acids in co-cultivation significantly increased, especially for oleic acid (32.45%) and linolenic acid (10.03%) compared with that in mono-cultivation. Moreover, high saturated and monounsaturated fatty acids (55.85%) were obtained in co-cultivation, which suggests their potential as a biodiesel feedstock. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Lipid productivity; Co-cultivation; Chlorella; Monoraphidium; Fatty acids; Biodiesel]
The scarcity of known petroleum reserves makes renewable energy resources highly attractive. The most feasible technique to meet the growing energy demand is the use of alternative fuels. An alternative fuel to petrodiesel must be technically feasible, economically competitive, environmentally acceptable, and easily available (1). One such fuel that exhibits great potential is biofuel, particularly biodiesel. Biodiesel (monoalkyl esters of fatty acids) is produced from vegetable oils, animal fats, restaurant waste and microalgal oils by transesterification or esterification with short chain alcohols. Although vegetable oils have been used as a diesel fuel source since the early 1930s, they may also be used for human consumption, thereby increasing the demand and cost for this resource because large areas of land, capital, and manpower are required for cultivation, which makes biodiesel production an economically challenging process (2,3). Although the use of animal fats and restaurant waste can reduce costs, their saturated compounds and crystallization at high temperatures are the disadvantages of using animal oils as a feedstock (4). Microalgae appear to be the only source of biodiesel with the potential to displace fossil diesel completely, and grow extremely rapidly, many are exceedingly rich in oil (3). However, production of biodiesel from microalgae is technically, but not yet economically, feasible (3). For the reduction of the production cost of biodiesel to compete with petrodiesel, high lipid productivity is a key desirable characteristic of species for biodiesel production, Microalgae should be simultaneously cultivated in low-
* Corresponding author at: School of Life Sciences, Yunnan Normal University, Kunming 650500, China. Tel.: þ86 0871 5920952; fax: þ86 0871 5920830. E-mail address: [email protected] (Z. Huang).
cost cultivation systems, such as co-cultivations. Co-cultivations are similar to mixed cultivations of microorganisms which are common in ecosystems, but with a unique difference in cultivation. In cocultivations the quantity and type of organisms in the cultivation are all defined at inoculation whereas in naturally occurring mixed cultivations, different organisms, depending on cultivation conditions, may become dominant during the cultivation period (5). The exploration of co-cultivation has become highly critical in many key biochemical processes. The benefits of this growth and lipid production strategy can potentially be exploited for high lipid productivity for biofuel. However, the reports on co-cultivation have mainly concentrated on the two different nutritional growth modes by addition of organic carbon source, and heterotrophic and mixotrophic nutrition. For example, co-cultivation of microalgae and yeast under heterotrophic condition showed improvements in cellular biomass and oil accumulation (6). A Louisiana native cocultivation of microalgae and cyanobacteria under mixotrophic condition using sodium acetate as carbon source resulted in high mean biomass productivity (7). In this study, microalgae Chlorella sp. U4341 and Monoraphidium sp. FXY-10 were isolated from a local lake, and used in co-cultivation under photoautotrophic condition without an organic carbon source to reduce the cost of raw materials for biofuel production. Lipid productivity and fatty acid composition were compared with those of the mono-cultivations. Both Chlorella and Monoraphidium are the most promising feedstock for biodiesel (8,9). Chlorella sp. U4341 has a relatively high growth rate and quick nutrient consumption ability. Monoraphidium sp. FXY-10 is an oleaginous microalga that can accumulate high amount of lipids especially when nutrition is low (10). Compared with other lipid-producing microalgae, Chlorella sp. U4341 and Monoraphidium sp. FXY-10 in a
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.12.014
Please cite this article in press as: Zhao, P., et al., Enhancing lipid productivity by co-cultivation of Chlorella sp. U4341 and Monoraphidium sp. FXY-10, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2013.12.014
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FIG. 1. Growth curves (A) and nitrate consumption profiles (B) of Chlorella sp. U4341 and Monoraphidium sp. FXY-10 in mono- and co-cultivation conditions. Bars are means of three replications SD.
co-cultivation system have the potential to accumulate more total lipids in a relatively short time. To our knowledge this study is the first to report on the lipid-accumulating properties of Chlorella and Monoraphidium sp. cells in co-cultivation. This study aimed to investigate a novel method for increasing lipid productivity and decreasing the cost of raw materials for biofuels production in a coculturing system.
Microorganisms and cultivation conditions Chlorella sp. U4341 and Monoraphidium sp. FXY-10 were isolated from water samples collected from Lake Fuxian (24 200 6700 N and 102 320 900 E), a plateau freshwater lake in Yunnan Province in China. U4341 and FXY-10 were cultivated using modified Bold’s Basal medium with the following composition: NaNO3 (1.5 g L1), KH2PO4 (0.7 g L1), K2HPO4∙3H2O (0.45 g L1), MgSO4∙7H2O (0.3 g L1), FeSO4∙7H2O (3 mg L1) and trace metal mix A5 (1 mL L1). All media were adjusted to pH 6.3 prior to autoclaving. Photoautotrophic flask cultivations of U4341, FXY-10, or a mixture of both were carried out in 200 mL Erlenmeyer flasks containing 500 mL of the cultivation medium autoclaved for 20 min at 121 C, and inoculated with exponentially growing microalgae. Thus, initial cell count of U4341 and FXY-10 were 3.41 106 and 4.26 106 cells mL1, respectively. The microalgae were continuously illuminated at 70 m Em2 s1 intensity, with white fluorescent light within the shaker and orbital shaking at 150 rpm at 25 1 C. No extra carbon dioxide was provided except what was naturally existing in the atmosphere to all the cultivations. Determination of algal growth and biomass concentrations To evaluate the growth of microalgae in mono- and co-cultivations, cell numbers were counted using a hemacytometer microscope (Nikon eclipse 50i). The specific growth rate (m) was calculated according to the following equation:
. tf ti
Lipid contentð%Þ ¼ WL =WA 100%
(2)
where WL (g) is the weight of the extracted lipids and WA (g) is the dry algae biomass. The lipid productivity was calculated as follows: PLipid mg l1 d1 ¼ WA ðgÞ CLipid ð%Þ=V ðlÞ TðdÞ
MATERIALS AND METHODS
m ¼ ln Nf ln Ni
twice. The collected extract was evaporated at 40 C, dried at 70 C for 2 h, and subsequently weighed after cooling to room temperature. The lipid content was calculated using the following equation:
(3)
where PLipid is the lipid productivity, CLipid is the lipid content, V is the working volume, and T is the cultivation time. Fatty acid composition analysis Fatty acid methyl esters (FAMEs) were prepared by in situ transesterification on lyophilized cells (13). The lipids were solubilized in 0.2 mL of chloroform/methanol (2:1, v/v), and simultaneously transesterified in situ with 0.3 mL of HCl/MeOH (5%, v/v) for 1 h at 85 C. The resulting FAMEs were extracted with 1 mL of n-hexane at room temperature for at least 1 h, and the top n-hexane layer was analyzed by gas chromatography/ mass spectrometry (Agilent 7890A/5975C) with an HP-5MS capillary column (5% phenyl methyl silox, 30 m 0.25 mm 0.25 mm). The injector was as 250 C with an injection of 1 mL under split mode (1:40). The temperature was programmed to 170e190 C at a rate of 10 C min1, 190 C (1 min) to 207 C at a rate of 0.8 C min1, and held at 207 C for 1 min. The carrier gas was helium, with a flow rate of 1.0 mL min1. The mass spectrometer was operated in EI (Electron Ionization) mode at 70 eV, and the scanned mass ranged from 15 amu to 650 amu. Peak identification was accomplished by comparing the mass spectra with the National Institute of Standards and Technology (NIST) mass spectral library (NIST08.L). Experiments were performed in triplicate, and data are expressed as mean standard deviation (SD). The degree of unsaturation (DU) was calculated as follows (14):
(1)
DU ¼ ðmonounsaturated; wt:%Þ þ 2 ðpolyunsaturated; wt:%Þ
where N is the cell density (cells mL1) at final (f) or initial (i) at time (t). Cells were harvested by centrifugation at 13,000 g for 5 min after cultivation. The pellets were washed twice with deionized water, frozen overnight at 70 C and freeze-dried at 80 C under vacuum conditions for 24 h. The pellets were weighed and considered as dry biomass weight (DBW). Biomass productivity was calculated by dividing the DBW with cultivation time (d). Experiments were performed in triplicate, and data are expressed as mean standard deviation (SD).
The long chain saturated factor (LCSF) was calculated as follows (14):
(4)
LCSF ¼ 0:1 C16ðwt:%Þ þ 0:5 C18ðwt:%Þ þ 1 C20ðwt:%Þ þ 1:5 C22ðwt:%Þ þ 2 C24ðwt:%Þ (5) The cold filter plugging point (CFPP) was calculated as follows (14):
Nitrate determination Nitrate concentration in medium was determined by a colorimetric method (11). In brief, 1 mL algae culture was collected and centrifuged, and 100 mL of the supernatant was mixed with 400 uL of 5% (w/v) salicylic acid in concentrated H2SO4. After incubation at room temperature for 20 min, 9.5 mL of 2 M NaOH was slowly added. Samples were cooled to room temperature, and absorbance was measured at 410 nm.
CFPP ¼ 3:1417 LCSF 16:477
Lipid extraction and determination Total lipid extraction from dry biomass was performed according to the procedure of Bligh and Dyer (12). The dry biomass was ground into a fine powder, 1 g of power was blended with 3 mL of chloroform/ methanol (2:1). The mixture was agitated for 20 min in an orbital shaker at 100 rpm at room temperature. The solvent phase was recovered by centrifugation at 2000 g for 10 min. The pellet was re-extracted in 3 mL of chloroform/methanol solution
Growth characteristics of the microalgae in mono- and cocultivations The dynamics of the growth curves of the three cultivations are shown in Fig. 1A. The logistic growth model gives the best fitting degree to experimental data, but U4341 in cocultivation use BiDoseResp function to show high degree of
(6)
RESULTS
Please cite this article in press as: Zhao, P., et al., Enhancing lipid productivity by co-cultivation of Chlorella sp. U4341 and Monoraphidium sp. FXY-10, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2013.12.014
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TABLE 1. Specific growth rate, biomass productivity, lipid content, and lipid productivity of Chlorella sp. U4341 and Monoraphidium sp. FXY-10 under mono- and coculture conditions. Parameters
m (d1)
U4341
FXY-10
U4341 þ FXY-10
0.26 0.05 0.15 0.03 U: 0.23 0.02 F: 0.07 0.00 1.46 0.57 0.89 0.22 1.24 0.36 58.4 0.57 35.60 0.22 62.00 0.36
Dry weight (g L1) Biomass productivity (mg L1 d1) Lipid content (%) 32.03 2.82 51.72 1.69 47.79 1.01 Lipid productivity 17.99 3.39 17.70 1.19 29.52 1.13 1 1 (mg L d ) Each data value represents the mean (SD) of three replications.
fitting. Although the growth pattern of U4341 in co-cultivation was similar to that in mono-cultivation, the growth rate of algae in cocultivation slightly decreased than that in mono-cultivation because of competition. At cultivation time of 26 d, the final cell densities of U4341 and FXY-10 were 47.2 106 cells mL1 and 29 106 cells mL1 in mono-cultivation, respectively, but 33.4 106 cells mL1 and 8.75 106 cells mL1 in co-cultivation at 20 d, respectively (Fig. 1A). Cell concentrations of FXY-10 in cocultivation slowly increased exponentially until day 10, after which growth rate began to decrease because the high density of U4341 suppressed the growth of FXY-10 over the entire experimental period. The cell numbers of U4341 and FXY-10 started to decrease after day 18, which may be caused by increasing flocculation tendency affecting accurate cell count measurements. As shown in Fig. 1B, the nitrogen source likely limited growth toward the end of the culture as the nitrate concentration by day 16(co-cultivation), day 22(U4341 in monocultivation), and day 26(FXY-10 in mono-cultivation) declined to almost nil. When nitrate in co-cultivation was almost depleted on day 16, the residual nitrates of U4341 and FXY-10 in monocultivations were approximately 16% and 47%, respectively. On day 20, these values were approximately 5% and 30%, respectively. The growth rates differed between the examined microalgae (Table 1). None of the cultivations exhibited a pronounced lag phase
3
because of an increase in the inoculum size of the desired algal species to overcome contamination from invading microorganisms. The mono-cultivation of U4341 grew faster than that of FXY-10, with growth rates of 0.26 and 0.15 day1, during the exponential growth phase. The growth rate of U4341 in co-cultivation (0.23 day1) was almost unaffected or slightly inhibited in monocultivation, but the rates of FXY-10 were 0.07 day1 in co-cultivation and 0.15 day1 in mono-cultivation. Biomass, lipid content, and lipid productivity Two microalgae were tested for biomass, lipid content and lipid production in 500 mL flask laboratory cultivations in mono- and co-cultivations under photoautotrophic condition. Table 1 shows that U4341 and FXY-10 in mono-cultivation had maximum and minimum dry weights (1.46 and 0.89 g L1) after cultivation, respectively. The biomass yield (1.24 g L1) of two algae in co-cultivation was similar to that in mono-cultivation. However, co-cultivation produced the highest biomass productivity (62.00 mg L1 d1) than mono-cultivations, which could be attributed to the shorter cultivation time. FXY-10 showed the highest lipid content (51.72%) with a low growth rate, whereas U4341 showed an opposite tendency in mono-cultivation. In this study, lipid productivity, which combines the dual effects of lipid content and biomass productivity, was used as a performance index to examine lipid production efficiency. The lipid productivity for U4341 and FXY-10 in co-cultivation was 29.52 mg L1 d1, which was significantly higher than those in mono-cultivation (17.99 and 17.70 mg L1 d1). Therefore, cocultivation is a novel method with great potential for biodiesel production, based on high lipid percentage and productivity. Fatty acid composition analysis Fatty acid composition of microalgae is also an important characteristic because it ultimately affects the quality of the biodiesel product. Depending on species or cultivation methods, microalgae produce many different kinds of lipids. Fatty acid biosynthesis from the biomass of U4341 and FXY10 in mono- and co-cultivation conditions were investigated and
TABLE 2. Comparison of the fatty acid profile of total fatty acids identified in Chlorella sp. U4341 and Monoraphidium sp. FXY-10 under mono- and co-culture conditions and vegetable oils. Fatty acids
C12:0 C14:0 C16:0 C16:1 C16:2 C16:3 C16:4 C18:0 C18:1 C18:2 C18:3 C18:4 C20:0 C20:1 C20:4 C22:0 C22:1 C24:0 Saturated Monounsaturated Polyunsaturated DU LCSF CFPP
Fatty acid content (% w/w) U4341
FXY-10
U4341 þ FXY-10
Palma
Rapea
Sunflowera
ND ND 23.04 1.5 ND 8.4 1.0 5.79 0.6 ND ND 21.93 1.5 21.9 1.7 4.27 0.4 ND ND ND ND ND ND ND 23.04 1.5 21.93 1.5 40.36 3.7 102.65 8.9 2.30 0.2 9.25 0.5
ND ND 30.7 2.4 ND ND 7.6 1.2 7.56 0.9 ND 21.64 1.8 5.36 0.6 2.39 0.2 13.76 1.1 ND ND ND ND ND ND 30.7 2.4 21.64 1.8 36.67 4.0 94.98 9.8 3.07 0.2 6.83 0.8
ND 4.43 0.5 14.62 1.2 1.01 0.2 0.55 0.0 0.5 0.1 0.26 0.0 3.34 0.5 32.45 2.2 18.62 1.4 10.03 1.1 0.78 0.1 ND ND 0.44 0.0 ND ND ND 22.39 2.2 33.46 2.4 31.18 2.7 95.82 7.8 3.13 0.4 6.64 1.2
0.1 0.7 36.7 0.1
0.0 0.0 4.9 0.0
0.0 0.0 6.2 0.1
6.6 46.1 8.6 0.3
1.6 33.0 20.4 7.9
3.7 25.2 63.1 0.2
0.4 0.2
0.0 9.3
0.3 0.2
0.1 0.0 0.1 44.7 46.4 8.9 64.2 7.7 10
0.0 23.0 0.0 6.5 65.3 28.3 121.9 1.3 10
0.7 0.1 0.2 11.1 25.6 63.3 152.2 4.2 3
ND, below the limit of detection. Blank indicates no information available. Each data value represents the mean (SD) of three replications. DU, degree of unsaturation; LCSF, long chain saturated factor; CFPP, cold filter plugging point. a Vegetable oils typically used to produce biodiesel (14).
Please cite this article in press as: Zhao, P., et al., Enhancing lipid productivity by co-cultivation of Chlorella sp. U4341 and Monoraphidium sp. FXY-10, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2013.12.014
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compared with vegetable oils typically used to produce biodiesel (Table 2). Oleic acid (18:1), palmitic acid (16:0), and linoleic acid (18:2) are major fatty acids. The two algae produced significantly higher content of oleic acid and linolenic acid (18:3) in co-cultivation than those in mono-cultivation. Palmitic acid remained high in monocultivations (23.04% and 30.7%) but significantly decreased in cocultivation (14.62%). The two algae possessed excellent capability of linoleic acid biosynthesis, with 21.9% in mono-cultivation and 18.62% in co-cultivation for U4341, but only 5.36 % for FXY-10 in mono-cultivation.
DISCUSSION Microalgae with high biomass productivity and lipid content are crucial for biodiesel production. However, the algal growth rate and lipid content are not necessarily correlated (17,18). Several microalgae species can be induced to accumulate substantial lipid quantities to obtain high oil yield. However, a high lipid content in algae can be achieved in case of a significant reduction in growth rate. For example, some algae have oil contents of 80% with extremely slow growth rates (3), and the algae of marine Tetraselmis suecica and freshwater Chlorophyta sorokiniana have very high growth rates and low lipid content (15). High lipid content is often offset by lower growth rates (16). The increase in lipid content dose not result in increased lipid productivity but leads to lower biomass and lipid productivity. Thus, recent studies have started to focus on lipid productivity for biodiesel production. As a novel strategy, the co-cultivation system can solve the contradiction between biomass productivity and lipid content to attain significantly higher lipid productivity. In co-cultivation, microalga (Chlorella) and bacterium (Azospirillum) (19), microalga (Chlorella vulgaris) and yeast (Rhodotorula glutinis) (20), and microalga (Spirulina platensis) and yeast (R. glutinis) (21) were enhanced to accelerate the accumulation of total biomass and total lipid yield. However, bacteria and yeast required an external source of organic compounds and nutrients as an energy source to increase the biodiesel production cost and biodiesel price. Cocultivation under photoautotrophic growth was a cost-effective technique that used freely available sunlight and inexpensive carbon dioxide. The lipid productivity (29.52 mg L1 d1) obtained in this study was higher than that in the co-cultivation of microalga (Isochrysis galbana 8701) and yeast (Ambrosiozyma cicatricose) (20.71 mg L1) (22) and in the co-cultivation of microalga (C. vulgaris) and cyanobacterium (Leptolyngbya sp.) (24.07 mg L1) (9). However, the lipid productivity (29.52 mg L1 d1) in this study was lower than those in previous reports (93.4e223.42 mg L1 d1) (21,23). This finding was possibly caused by not adding organic carbon source in this study (photoautotrophic condition), compared with that used in the previous reports (heterotrophic or mixotrophic condition). In this study, co-cultivation of microalgae obtained higher lipid productivity than mono-cultivation because of the enhancement in total lipid production and reduction in incubation time. The synergistic effects between two microalgae and nutrient-starvation conditions may be related to the increased lipids yield in co-cultivation conditions. It was reported that microalgae released more free fatty acids into media in co-cultivation conditions than that in normal culture conditions (24e26). The increased free fatty acids, key precursors in the biosynthesis of lipids, may stimulate the transcription of downstream genes involved in lipid biosynthesis (27,28). Besides, nutrient-starvation, especially nitrogen starvation was more likely to occur in co-cultivation conditions. However, nitrogen starvation may increase the intracellular content of acetylCoA, which is a major element required for the biosynthesis of
triglycerides, mainly neutral lipids (29,30). Thus, total lipid production was improved in a co-culture system. Nitrogen starvation might appear early or easily in microalgae in a co-culture system. Our data show that nitrate consumption in co-cultivation condition was significantly stronger than that in mono-cultivation conditions (Fig. 1B). It was possible that microalgae could be induced by cocultivation conditions to produce particularly high concentrations of chemical compounds such as peptides, amino acids and nitric oxide (31). Therefore, lipid accumulation of microalgae U4341 and FXY-10 were both triggered under nitrogen depleted co-cultivation condition, and the total lipid production subsequently improved. A similar conclusion was obtained from the experimental results. We calculated the dry weights of U4341 and FXY-10 to be approximately 1.03 and 0.27 (g L1) in co-cultivation, according to the proportion of cell density (Fig. 1A) and relationship of dry weight (Table 1). Lipid production values of U4341, FXY-10, and cocultivation were 467.7, 460.3 and 590.4 (mg L1), respectively. We determined which algal improves lipid production in co-cultivation. If the lipid content (32.03%) of U4341 was unchanged, then its lipid production was possibly 330 (mg L1) in mono-culture conditions. The biomass of FXY-10 (approximately 0.27 g L1) accumulated lipid production of 260.4 (mg L1). However, the lipid content of FXY-10 could be increased from 51.72% (mono-cultivation) to 96% (co-cultivation) to compensate the shortage of lipid production, which was not accomplished in the microalgae culture. The hypothesis of poison was rejected. Thus, the two microalgae contributed in improving lipid production. The culture time was shortened in co-culture condition (20 d vs. 26 d) because nitrogen consumption and the lipid accumulation were faster in co-cultivation. Therefore, biomass productivity and lipid productivity significantly increased (62 and 29.52 mg L1 d1) by shortening the culture time in co-cultivation. CO2 also greatly effects microalgae growth and lipid accumulation (32). Future investigations that will take advantage of this discovery should regulate the CO2 concentration in co-cultivation conditions to enhance lipid productivity. C. vulgaris Beijerink was first observed in algae to release substances affecting the growth of other organisms (33). Further investigations revealed that these substances were not a single compound but a mixture of fatty acids (34). The mixture was mainly composed of stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), and palmitic acid (C16:0) (24,35). Chiang et al. (35) observed the highest toxicity with linolenic acid, followed by linoleic, oleic, and palmitic acids. We obtained similar results in this experiment. The two algae grown in co-cultivation possessed significantly higher contents of oleic, linolenic, and linoleic acids than those in mono-cultivation. Thus, algal U4341 was forced in producing more C18 fatty acids, especially linolenic acid, to inhibit the growth of FXY-10 in co-cultivation under competition pressure, than that in mono-cultivation with relatively free cultivation circumstances. However, FXY-10 also generated fatty acids as competitive arms but exhibited low tolerance capacity, which resulted in growth suppression. By contrast, U4341 could strongly tolerate the toxicity against high concentration of C18 fatty acids, and growth was almost unchanged or only slightly inhibited. Not all algal oils are satisfactory for biodiesel production, but suitable oils do occur commonly. The fatty acid profiles of cocultivation in this study were mainly 16 to 18 carbon atoms, which are suitable for biodiesel production (2). The lipid quality analyzed in both species suggests that a biodiesel derived from these oils may present an acceptable cetane number (CN), iodine value (IV), and CFPP (36). The DU of the oil defined by Ramos et al. (14) is an important parameter in determining the CN and IV of the final biodiesel product. The DU for the algal oil extracted from the biomass in co-cultivation was 95.82 (Table 2), which was lower
Please cite this article in press as: Zhao, P., et al., Enhancing lipid productivity by co-cultivation of Chlorella sp. U4341 and Monoraphidium sp. FXY-10, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2013.12.014
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than those of U4341 (102.65), oils of vegetable rape (121.9), sunflower (152.2) (14), and the microalgae Ankistrodesmus fusiformis (102.91), Kirchneriella lunaris (112.77), Botryococcus braunii (100.69) (37), and Auxenochlorella protothecoides (113.5) (38). Oils that have DU higher than 137 do not meet the European biodiesel standard for CN and IV (UNE-EN 14214) (38). However, the low percentage of polyunsaturated FAME (lower DU) composes a group that is more suitable for generating biodiesel with shorter ignition time (higher CN), less deposit formation, and better lubricity (lower IV), and produces better methyl ester fuels with higher oxidation stability (37,38). Although palm oil obtained a lower DU value (64.2) (14) than co-cultivation in this study, it had the poorest result of CFPP (10 C) (Table 2). CFPP is usually used to predict the flow performance of biodiesel at low temperatures (39). The higher will be the value of CFPP, and the worse their low temperature properties (14). The EN 14214 (biodiesel standard of UK) and ASTM D6751-07a (biodiesel standard of US) both do not mention a low-temperature parameter in their list of specifications, but CSN 65 6507 (biodiesel standard of Czech) and SS 155436 (biodiesel standard of Sweden) both specify temperature limits (5 C) to the CFPP. If liquid biodiesel is cooled, palm biodiesel with high methyl esters content of stearic and palmitic acid will typically constitute a major share of material recovered from clogged biodiesel fuel filters (40). The biodiesel of microalgae in co-cultivation had better CFPP values (6.64 C), which implies good low-temperature filterability. Thus, U4341 and FXY-10 in co-cultivation could be recognized as good lipid producers for biodiesel based on the values of DU and CFPP. ACKNOWLEDGMENTS This research was supported by the National High Technology Research and Development Program of China (863 Program; no. 2008AA02Z202), the National Natural Science Foundation of China (no. 31160229) to Z. Huang and the National Natural Science Foundation of China (no. 21266013) to X. Yu. References 1. Lang, X., Dalaia, A. K., Bakhshia, N. N., Reaneyb, M. J., and Hertz, P. B.: Preparation and characterization of bio-diesels from various bio-oils, Bioresour. Technol., 80, 53e62 (2001). 2. Huang, G. H., Chen, F., Wei, D., Zhang, X. W., and Chen, G.: Biodiesel production by microalgal biotechnology, Appl. Energ., 87, 38e46 (2010). 3. Chisti, Y.: Biodiesel from microalgae, Biotechnol. Adv., 25, 294e306 (2007). 4. Alptekin, E. and Canakci, M.: Optimization of pretreatment reaction for methyl ester production from chicken fat, Fuel, 89, 4035e4039 (2010). 5. Oncel, S. S., Imamoglu, E., Gunerken, E., and Sukan, F. V.: Comparison of different cultivation modes and light intensities using mono-cultivations and co-cultivations of Haematococcus pluvialis and Chlorella zofingiensis, J. Chem. Technol. Biot., 86, 414e420 (2011). 6. Shu, C. H., Tsai, C. C., Chen, K. Y., Liao, W. H., and Huang, H. C.: Enhancing high quality oil accumulation and carbon dioxide fixation by a mixed culture of Chlorella sp. and Saccharomyces cerevisiae, J. Taiwan Inst. Chem. Eng., 44, 936e942 (2013). 7. Bai, R. and Gutierrez, M. T.: Effect of organic carbon, C:N ratio and light on the growth and lipid productivity of microalgae/cyanobacteria co-culture, Eng. Life Sci., 14, 47e56 (2014). 8. Liang, Y., Sarkany, N., and Cui, Y.: Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions, Biotechnol. Lett., 31, 1043e1049 (2009). 9. Bogen, C., Klassen, V., Wichmann, J., Russa, M. L., Doebbe, A., Grundmann, M., Uronen, P., Kruse, O., and Mussgnug, J. H.: Identification of Monoraphidium contortum as a promising species for liquid biofuel production, Bioresour. Technol., 133, 622e626 (2013). 10. Yu, X., Zhao, P., He, C., Li, J. J., Tang, X. H., Zhou, J. P., and Huang, Z. X.: Isolation of a novel strain of Monoraphidium sp. and characterization of its potential application as biodiesel feedstock, Bioresour. Technol., 121, 256e262 (2012). 11. Hecht, U. and Mohr, H.: Factors controlling nitrate and ammonium accumulation in mustard (Sinapis alba) seedlings, Physiol. Plant., 78, 379e387 (1990).
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12. Bligh, E. G. and Dyer, W. J.: A rapid method of total lipid extraction and purification, Can. J. Biochem. Physiol., 37, 911e917 (1959). 13. Laurens, L. M. L., Quinn, M., Wychen, S. V., Templeton, D. W., and Wolfrum, E. J.: Accurate and reliable quantification of total microalgal fuel potential as fatty acid methyl esters by in situ transesterification, Anal. Bioanal. Chem., 403, 167e178 (2012). 14. Ramos, M. J., Fernández, C. M., Casas, A., Rodríguez, L., and Pérez, A.: Influence of fatty acid composition of raw materials on biodiesel properties, Bioresour. Technol., 100, 261e268 (2009). 15. Griffiths, M. J. and Harrison, S. T. L.: Lipid productivity as a key characteristic for choosing algal species for biodiesel production, J. Appl. Phycol., 21, 493e507 (2009). 16. Huerlimann, R., Nys, R. D., and Heimann, K.: Growth, lipid content, productivity, and fatty acid composition of tropical microalgae for scale-up production, Biotechnol. Bioeng., 107, 245e257 (2010). 17. Rodolfi, L., Zittelli, G. C., Bassi, N., Padovani, G., Biondi, N., Bonini, G., and Tredici, M. R.: Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor, Biotechnol. Bioeng., 102, 100e112 (2008). 18. Sheehan, J., Dunahay, T., Benemann, J., and Roessler, P.: A look back at the U.S. Department of Energy’s Aquatic Species Program-biodiesel from algae. National Renewable Energy Laboratory, Golden (1998). 19. de-Bashan, L. E., Bashan, Y., Moreno, M., Lebsky, V. K., and Bustillos, J. J.: Increased pigment and lipid content, lipid variety, and cell and population size of the microalgae Chlorella spp. when co-immobilized in alginate beads with the microalgae-growth-promoting bacterium Azospirillum brasilense, Can. J. Microbiol., 48, 514e521 (2002). 20. Cheirsilp, B., Suwannarat, W., and Niyomdecha, R.: Mixed cultivation of oleaginous yeast Rhodotorula glutinis and microalga Chlorella vulgaris for lipid production from industrial wastes and its use as biodiesel feedstock, New Biotechnol., 28, 362e368 (2011). 21. Xue, F. Y., Miao, J. X., Zhang, X., and Tan, T. W.: A new strategy for lipids production by mix cultivation of Spirulina platensis and Rhodotorula glutinis, Appl. Biochem. Biotechnol., 160, 498e503 (2010). 22. Cai, S. Q., Hu, C. Q., and Du, S. B.: Comparisons of growth and biochemical composition between mixed culture of alga and yeast and monocultures, J. Biosci. Bioeng., 104, 391e397 (2007). 23. Papone, T., Kookkhunthod, S., and Leesing, R.: Microbial oil production by monoculture and mixed cultures of microalgae and oleaginous yeasts using sugarcane juice as substrate, World Acad. Sci. Eng. Technol., 64, 1127e1131 (2012). 24. Greca, M. D., Zarrelli, A., Fergola, P., Cerasuolo, M., Pollio, A., and Pinto, G.: Fatty acids released by Chlorella vulgaris and their role in interference with Pseudokirchneriella subcapitata: experiments and modelling, J. Chem. Ecol., 36, 339e349 (2010). 25. Tate, J. J., Wing, M. T. G., Rusch, K. A., and Benton, M. G.: The effects of plant growth substances and mixed cultures on growth and metabolite production of green algae Chlorella sp.: a review, J. Plant Growth Regul., 32, 417e428 (2013). 26. Fergola, P., Cerasuolo, M., Pollio, A., Pinto, G., and DellaGreca, M.: Allelopathy and competition between Chlorella vulgaris and Pseudokirchneriella subcapitata: experiments and mathematical model, Ecol. Model., 208, 205e214 (2007). 27. Ohlrogge, J. B. and Jaworski, J. G.: Regulation of fatty acid synthesis, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48, 109e136 (1997). 28. Coleman, R. A., Lewin, T. M., Van Horn, C. G., and Gonzalez- Baró, M. R.: Do long-chain acyl-CoA synthetases regulate fatty acid entry into synthetic versus degradative pathways? J. Nutr., 132, 2123e2126 (2002). 29. Sukenik, A. and Livne, A.: Variations in lipid and fatty acid content in relation to acetyl CoA carboxylase in the marine prymnesiophyte Isochrysis galbana, Plant. Cell. Physiol., 32, 371e378 (1991). 30. Takagi, M., Watanabe, K., Yamaberi, K., and Yoshida, T.: Limited feeding of potassium nitrate for intracellular lipid and triglyceride accumulation of Nannochloris sp. UTEX LB1999, Appl. Microbiol. Biotechnol., 54, 112e117 (2000). 31. Mendes, L. B. B. and Vermelho, A. B.: Allelopathy as a potential strategy to improve microalgae cultivation, Biotechnol. Biofuels, 6, 152e166 (2013). 32. Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., and Darzins, A.: Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances, Plant J., 54, 621e639 (2008). 33. Pratt, R. and Fong, J.: Studies on Chlorella vulgaris II. Further evidence that Chlorella cells form a growth-inhibiting substance, Am. J Bot., 27, 431e436 (1940). 34. Spoehr, H. A., Smith, J. H. C., Strain, H. H., Milner, H. W., and Hardin, G. J.: Fatty acids antibacterials from plants. Literary Licensing, LLC, Montana, USA (1949). 35. Chiang, I. Z., Huang, W. Y., and Wu, J. T.: Allelochemicals of Botryococcus braunii (Chlorophyceae), J. Phycol., 40, 474e480 (2004). 36. Popovich, C. A., Damiani, C., Consten, D., and Leonardi, P. I.: Lipid quality of the diatoms and from the South Atlantic Coast (Argentina):
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Please cite this article in press as: Zhao, P., et al., Enhancing lipid productivity by co-cultivation of Chlorella sp. U4341 and Monoraphidium sp. FXY-10, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2013.12.014
Enhancing lipid productivity by co-cultivation of Chlorella sp. U4341 and Monoraphidium sp. FXY-10 Peng Zhao,1, 2 Xuya Yu,1, 2 Junjun Li,1 Xianhua Tang,1 and Zunxi Huang1, 3, * Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Kunming 650500, China,1 College of Life Sciences and Biotechnology, Kunming University of Science and Technology, Kunming 650500, China,2 and School of Life Sciences, Yunnan Normal University, Kunming 650500, China3 Received 9 September 2013; accepted 18 December 2013 Available online xxx
To improve lipid productivity, co-cultivation of Chlorella sp. U4341 and Monoraphidium sp. FXY-10 for lipid production was studied. Compared with mono-cultivations, co-cultivation of the two microalgae significantly increased the accumulation of total biomass and total lipid yield, and enhanced the lipid productivity (29.52 mg LL1 dL1). Fatty acid compositions significantly varied in different cultivations. The content of C18 fatty acids in co-cultivation significantly increased, especially for oleic acid (32.45%) and linolenic acid (10.03%) compared with that in mono-cultivation. Moreover, high saturated and monounsaturated fatty acids (55.85%) were obtained in co-cultivation, which suggests their potential as a biodiesel feedstock. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Lipid productivity; Co-cultivation; Chlorella; Monoraphidium; Fatty acids; Biodiesel]
The scarcity of known petroleum reserves makes renewable energy resources highly attractive. The most feasible technique to meet the growing energy demand is the use of alternative fuels. An alternative fuel to petrodiesel must be technically feasible, economically competitive, environmentally acceptable, and easily available (1). One such fuel that exhibits great potential is biofuel, particularly biodiesel. Biodiesel (monoalkyl esters of fatty acids) is produced from vegetable oils, animal fats, restaurant waste and microalgal oils by transesterification or esterification with short chain alcohols. Although vegetable oils have been used as a diesel fuel source since the early 1930s, they may also be used for human consumption, thereby increasing the demand and cost for this resource because large areas of land, capital, and manpower are required for cultivation, which makes biodiesel production an economically challenging process (2,3). Although the use of animal fats and restaurant waste can reduce costs, their saturated compounds and crystallization at high temperatures are the disadvantages of using animal oils as a feedstock (4). Microalgae appear to be the only source of biodiesel with the potential to displace fossil diesel completely, and grow extremely rapidly, many are exceedingly rich in oil (3). However, production of biodiesel from microalgae is technically, but not yet economically, feasible (3). For the reduction of the production cost of biodiesel to compete with petrodiesel, high lipid productivity is a key desirable characteristic of species for biodiesel production, Microalgae should be simultaneously cultivated in low-
* Corresponding author at: School of Life Sciences, Yunnan Normal University, Kunming 650500, China. Tel.: þ86 0871 5920952; fax: þ86 0871 5920830. E-mail address: [email protected] (Z. Huang).
cost cultivation systems, such as co-cultivations. Co-cultivations are similar to mixed cultivations of microorganisms which are common in ecosystems, but with a unique difference in cultivation. In cocultivations the quantity and type of organisms in the cultivation are all defined at inoculation whereas in naturally occurring mixed cultivations, different organisms, depending on cultivation conditions, may become dominant during the cultivation period (5). The exploration of co-cultivation has become highly critical in many key biochemical processes. The benefits of this growth and lipid production strategy can potentially be exploited for high lipid productivity for biofuel. However, the reports on co-cultivation have mainly concentrated on the two different nutritional growth modes by addition of organic carbon source, and heterotrophic and mixotrophic nutrition. For example, co-cultivation of microalgae and yeast under heterotrophic condition showed improvements in cellular biomass and oil accumulation (6). A Louisiana native cocultivation of microalgae and cyanobacteria under mixotrophic condition using sodium acetate as carbon source resulted in high mean biomass productivity (7). In this study, microalgae Chlorella sp. U4341 and Monoraphidium sp. FXY-10 were isolated from a local lake, and used in co-cultivation under photoautotrophic condition without an organic carbon source to reduce the cost of raw materials for biofuel production. Lipid productivity and fatty acid composition were compared with those of the mono-cultivations. Both Chlorella and Monoraphidium are the most promising feedstock for biodiesel (8,9). Chlorella sp. U4341 has a relatively high growth rate and quick nutrient consumption ability. Monoraphidium sp. FXY-10 is an oleaginous microalga that can accumulate high amount of lipids especially when nutrition is low (10). Compared with other lipid-producing microalgae, Chlorella sp. U4341 and Monoraphidium sp. FXY-10 in a
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.12.014
Please cite this article in press as: Zhao, P., et al., Enhancing lipid productivity by co-cultivation of Chlorella sp. U4341 and Monoraphidium sp. FXY-10, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2013.12.014
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J. BIOSCI. BIOENG.,
FIG. 1. Growth curves (A) and nitrate consumption profiles (B) of Chlorella sp. U4341 and Monoraphidium sp. FXY-10 in mono- and co-cultivation conditions. Bars are means of three replications SD.
co-cultivation system have the potential to accumulate more total lipids in a relatively short time. To our knowledge this study is the first to report on the lipid-accumulating properties of Chlorella and Monoraphidium sp. cells in co-cultivation. This study aimed to investigate a novel method for increasing lipid productivity and decreasing the cost of raw materials for biofuels production in a coculturing system.
Microorganisms and cultivation conditions Chlorella sp. U4341 and Monoraphidium sp. FXY-10 were isolated from water samples collected from Lake Fuxian (24 200 6700 N and 102 320 900 E), a plateau freshwater lake in Yunnan Province in China. U4341 and FXY-10 were cultivated using modified Bold’s Basal medium with the following composition: NaNO3 (1.5 g L1), KH2PO4 (0.7 g L1), K2HPO4∙3H2O (0.45 g L1), MgSO4∙7H2O (0.3 g L1), FeSO4∙7H2O (3 mg L1) and trace metal mix A5 (1 mL L1). All media were adjusted to pH 6.3 prior to autoclaving. Photoautotrophic flask cultivations of U4341, FXY-10, or a mixture of both were carried out in 200 mL Erlenmeyer flasks containing 500 mL of the cultivation medium autoclaved for 20 min at 121 C, and inoculated with exponentially growing microalgae. Thus, initial cell count of U4341 and FXY-10 were 3.41 106 and 4.26 106 cells mL1, respectively. The microalgae were continuously illuminated at 70 m Em2 s1 intensity, with white fluorescent light within the shaker and orbital shaking at 150 rpm at 25 1 C. No extra carbon dioxide was provided except what was naturally existing in the atmosphere to all the cultivations. Determination of algal growth and biomass concentrations To evaluate the growth of microalgae in mono- and co-cultivations, cell numbers were counted using a hemacytometer microscope (Nikon eclipse 50i). The specific growth rate (m) was calculated according to the following equation:
. tf ti
Lipid contentð%Þ ¼ WL =WA 100%
(2)
where WL (g) is the weight of the extracted lipids and WA (g) is the dry algae biomass. The lipid productivity was calculated as follows: PLipid mg l1 d1 ¼ WA ðgÞ CLipid ð%Þ=V ðlÞ TðdÞ
MATERIALS AND METHODS
m ¼ ln Nf ln Ni
twice. The collected extract was evaporated at 40 C, dried at 70 C for 2 h, and subsequently weighed after cooling to room temperature. The lipid content was calculated using the following equation:
(3)
where PLipid is the lipid productivity, CLipid is the lipid content, V is the working volume, and T is the cultivation time. Fatty acid composition analysis Fatty acid methyl esters (FAMEs) were prepared by in situ transesterification on lyophilized cells (13). The lipids were solubilized in 0.2 mL of chloroform/methanol (2:1, v/v), and simultaneously transesterified in situ with 0.3 mL of HCl/MeOH (5%, v/v) for 1 h at 85 C. The resulting FAMEs were extracted with 1 mL of n-hexane at room temperature for at least 1 h, and the top n-hexane layer was analyzed by gas chromatography/ mass spectrometry (Agilent 7890A/5975C) with an HP-5MS capillary column (5% phenyl methyl silox, 30 m 0.25 mm 0.25 mm). The injector was as 250 C with an injection of 1 mL under split mode (1:40). The temperature was programmed to 170e190 C at a rate of 10 C min1, 190 C (1 min) to 207 C at a rate of 0.8 C min1, and held at 207 C for 1 min. The carrier gas was helium, with a flow rate of 1.0 mL min1. The mass spectrometer was operated in EI (Electron Ionization) mode at 70 eV, and the scanned mass ranged from 15 amu to 650 amu. Peak identification was accomplished by comparing the mass spectra with the National Institute of Standards and Technology (NIST) mass spectral library (NIST08.L). Experiments were performed in triplicate, and data are expressed as mean standard deviation (SD). The degree of unsaturation (DU) was calculated as follows (14):
(1)
DU ¼ ðmonounsaturated; wt:%Þ þ 2 ðpolyunsaturated; wt:%Þ
where N is the cell density (cells mL1) at final (f) or initial (i) at time (t). Cells were harvested by centrifugation at 13,000 g for 5 min after cultivation. The pellets were washed twice with deionized water, frozen overnight at 70 C and freeze-dried at 80 C under vacuum conditions for 24 h. The pellets were weighed and considered as dry biomass weight (DBW). Biomass productivity was calculated by dividing the DBW with cultivation time (d). Experiments were performed in triplicate, and data are expressed as mean standard deviation (SD).
The long chain saturated factor (LCSF) was calculated as follows (14):
(4)
LCSF ¼ 0:1 C16ðwt:%Þ þ 0:5 C18ðwt:%Þ þ 1 C20ðwt:%Þ þ 1:5 C22ðwt:%Þ þ 2 C24ðwt:%Þ (5) The cold filter plugging point (CFPP) was calculated as follows (14):
Nitrate determination Nitrate concentration in medium was determined by a colorimetric method (11). In brief, 1 mL algae culture was collected and centrifuged, and 100 mL of the supernatant was mixed with 400 uL of 5% (w/v) salicylic acid in concentrated H2SO4. After incubation at room temperature for 20 min, 9.5 mL of 2 M NaOH was slowly added. Samples were cooled to room temperature, and absorbance was measured at 410 nm.
CFPP ¼ 3:1417 LCSF 16:477
Lipid extraction and determination Total lipid extraction from dry biomass was performed according to the procedure of Bligh and Dyer (12). The dry biomass was ground into a fine powder, 1 g of power was blended with 3 mL of chloroform/ methanol (2:1). The mixture was agitated for 20 min in an orbital shaker at 100 rpm at room temperature. The solvent phase was recovered by centrifugation at 2000 g for 10 min. The pellet was re-extracted in 3 mL of chloroform/methanol solution
Growth characteristics of the microalgae in mono- and cocultivations The dynamics of the growth curves of the three cultivations are shown in Fig. 1A. The logistic growth model gives the best fitting degree to experimental data, but U4341 in cocultivation use BiDoseResp function to show high degree of
(6)
RESULTS
Please cite this article in press as: Zhao, P., et al., Enhancing lipid productivity by co-cultivation of Chlorella sp. U4341 and Monoraphidium sp. FXY-10, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2013.12.014
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TABLE 1. Specific growth rate, biomass productivity, lipid content, and lipid productivity of Chlorella sp. U4341 and Monoraphidium sp. FXY-10 under mono- and coculture conditions. Parameters
m (d1)
U4341
FXY-10
U4341 þ FXY-10
0.26 0.05 0.15 0.03 U: 0.23 0.02 F: 0.07 0.00 1.46 0.57 0.89 0.22 1.24 0.36 58.4 0.57 35.60 0.22 62.00 0.36
Dry weight (g L1) Biomass productivity (mg L1 d1) Lipid content (%) 32.03 2.82 51.72 1.69 47.79 1.01 Lipid productivity 17.99 3.39 17.70 1.19 29.52 1.13 1 1 (mg L d ) Each data value represents the mean (SD) of three replications.
fitting. Although the growth pattern of U4341 in co-cultivation was similar to that in mono-cultivation, the growth rate of algae in cocultivation slightly decreased than that in mono-cultivation because of competition. At cultivation time of 26 d, the final cell densities of U4341 and FXY-10 were 47.2 106 cells mL1 and 29 106 cells mL1 in mono-cultivation, respectively, but 33.4 106 cells mL1 and 8.75 106 cells mL1 in co-cultivation at 20 d, respectively (Fig. 1A). Cell concentrations of FXY-10 in cocultivation slowly increased exponentially until day 10, after which growth rate began to decrease because the high density of U4341 suppressed the growth of FXY-10 over the entire experimental period. The cell numbers of U4341 and FXY-10 started to decrease after day 18, which may be caused by increasing flocculation tendency affecting accurate cell count measurements. As shown in Fig. 1B, the nitrogen source likely limited growth toward the end of the culture as the nitrate concentration by day 16(co-cultivation), day 22(U4341 in monocultivation), and day 26(FXY-10 in mono-cultivation) declined to almost nil. When nitrate in co-cultivation was almost depleted on day 16, the residual nitrates of U4341 and FXY-10 in monocultivations were approximately 16% and 47%, respectively. On day 20, these values were approximately 5% and 30%, respectively. The growth rates differed between the examined microalgae (Table 1). None of the cultivations exhibited a pronounced lag phase
3
because of an increase in the inoculum size of the desired algal species to overcome contamination from invading microorganisms. The mono-cultivation of U4341 grew faster than that of FXY-10, with growth rates of 0.26 and 0.15 day1, during the exponential growth phase. The growth rate of U4341 in co-cultivation (0.23 day1) was almost unaffected or slightly inhibited in monocultivation, but the rates of FXY-10 were 0.07 day1 in co-cultivation and 0.15 day1 in mono-cultivation. Biomass, lipid content, and lipid productivity Two microalgae were tested for biomass, lipid content and lipid production in 500 mL flask laboratory cultivations in mono- and co-cultivations under photoautotrophic condition. Table 1 shows that U4341 and FXY-10 in mono-cultivation had maximum and minimum dry weights (1.46 and 0.89 g L1) after cultivation, respectively. The biomass yield (1.24 g L1) of two algae in co-cultivation was similar to that in mono-cultivation. However, co-cultivation produced the highest biomass productivity (62.00 mg L1 d1) than mono-cultivations, which could be attributed to the shorter cultivation time. FXY-10 showed the highest lipid content (51.72%) with a low growth rate, whereas U4341 showed an opposite tendency in mono-cultivation. In this study, lipid productivity, which combines the dual effects of lipid content and biomass productivity, was used as a performance index to examine lipid production efficiency. The lipid productivity for U4341 and FXY-10 in co-cultivation was 29.52 mg L1 d1, which was significantly higher than those in mono-cultivation (17.99 and 17.70 mg L1 d1). Therefore, cocultivation is a novel method with great potential for biodiesel production, based on high lipid percentage and productivity. Fatty acid composition analysis Fatty acid composition of microalgae is also an important characteristic because it ultimately affects the quality of the biodiesel product. Depending on species or cultivation methods, microalgae produce many different kinds of lipids. Fatty acid biosynthesis from the biomass of U4341 and FXY10 in mono- and co-cultivation conditions were investigated and
TABLE 2. Comparison of the fatty acid profile of total fatty acids identified in Chlorella sp. U4341 and Monoraphidium sp. FXY-10 under mono- and co-culture conditions and vegetable oils. Fatty acids
C12:0 C14:0 C16:0 C16:1 C16:2 C16:3 C16:4 C18:0 C18:1 C18:2 C18:3 C18:4 C20:0 C20:1 C20:4 C22:0 C22:1 C24:0 Saturated Monounsaturated Polyunsaturated DU LCSF CFPP
Fatty acid content (% w/w) U4341
FXY-10
U4341 þ FXY-10
Palma
Rapea
Sunflowera
ND ND 23.04 1.5 ND 8.4 1.0 5.79 0.6 ND ND 21.93 1.5 21.9 1.7 4.27 0.4 ND ND ND ND ND ND ND 23.04 1.5 21.93 1.5 40.36 3.7 102.65 8.9 2.30 0.2 9.25 0.5
ND ND 30.7 2.4 ND ND 7.6 1.2 7.56 0.9 ND 21.64 1.8 5.36 0.6 2.39 0.2 13.76 1.1 ND ND ND ND ND ND 30.7 2.4 21.64 1.8 36.67 4.0 94.98 9.8 3.07 0.2 6.83 0.8
ND 4.43 0.5 14.62 1.2 1.01 0.2 0.55 0.0 0.5 0.1 0.26 0.0 3.34 0.5 32.45 2.2 18.62 1.4 10.03 1.1 0.78 0.1 ND ND 0.44 0.0 ND ND ND 22.39 2.2 33.46 2.4 31.18 2.7 95.82 7.8 3.13 0.4 6.64 1.2
0.1 0.7 36.7 0.1
0.0 0.0 4.9 0.0
0.0 0.0 6.2 0.1
6.6 46.1 8.6 0.3
1.6 33.0 20.4 7.9
3.7 25.2 63.1 0.2
0.4 0.2
0.0 9.3
0.3 0.2
0.1 0.0 0.1 44.7 46.4 8.9 64.2 7.7 10
0.0 23.0 0.0 6.5 65.3 28.3 121.9 1.3 10
0.7 0.1 0.2 11.1 25.6 63.3 152.2 4.2 3
ND, below the limit of detection. Blank indicates no information available. Each data value represents the mean (SD) of three replications. DU, degree of unsaturation; LCSF, long chain saturated factor; CFPP, cold filter plugging point. a Vegetable oils typically used to produce biodiesel (14).
Please cite this article in press as: Zhao, P., et al., Enhancing lipid productivity by co-cultivation of Chlorella sp. U4341 and Monoraphidium sp. FXY-10, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2013.12.014
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compared with vegetable oils typically used to produce biodiesel (Table 2). Oleic acid (18:1), palmitic acid (16:0), and linoleic acid (18:2) are major fatty acids. The two algae produced significantly higher content of oleic acid and linolenic acid (18:3) in co-cultivation than those in mono-cultivation. Palmitic acid remained high in monocultivations (23.04% and 30.7%) but significantly decreased in cocultivation (14.62%). The two algae possessed excellent capability of linoleic acid biosynthesis, with 21.9% in mono-cultivation and 18.62% in co-cultivation for U4341, but only 5.36 % for FXY-10 in mono-cultivation.
DISCUSSION Microalgae with high biomass productivity and lipid content are crucial for biodiesel production. However, the algal growth rate and lipid content are not necessarily correlated (17,18). Several microalgae species can be induced to accumulate substantial lipid quantities to obtain high oil yield. However, a high lipid content in algae can be achieved in case of a significant reduction in growth rate. For example, some algae have oil contents of 80% with extremely slow growth rates (3), and the algae of marine Tetraselmis suecica and freshwater Chlorophyta sorokiniana have very high growth rates and low lipid content (15). High lipid content is often offset by lower growth rates (16). The increase in lipid content dose not result in increased lipid productivity but leads to lower biomass and lipid productivity. Thus, recent studies have started to focus on lipid productivity for biodiesel production. As a novel strategy, the co-cultivation system can solve the contradiction between biomass productivity and lipid content to attain significantly higher lipid productivity. In co-cultivation, microalga (Chlorella) and bacterium (Azospirillum) (19), microalga (Chlorella vulgaris) and yeast (Rhodotorula glutinis) (20), and microalga (Spirulina platensis) and yeast (R. glutinis) (21) were enhanced to accelerate the accumulation of total biomass and total lipid yield. However, bacteria and yeast required an external source of organic compounds and nutrients as an energy source to increase the biodiesel production cost and biodiesel price. Cocultivation under photoautotrophic growth was a cost-effective technique that used freely available sunlight and inexpensive carbon dioxide. The lipid productivity (29.52 mg L1 d1) obtained in this study was higher than that in the co-cultivation of microalga (Isochrysis galbana 8701) and yeast (Ambrosiozyma cicatricose) (20.71 mg L1) (22) and in the co-cultivation of microalga (C. vulgaris) and cyanobacterium (Leptolyngbya sp.) (24.07 mg L1) (9). However, the lipid productivity (29.52 mg L1 d1) in this study was lower than those in previous reports (93.4e223.42 mg L1 d1) (21,23). This finding was possibly caused by not adding organic carbon source in this study (photoautotrophic condition), compared with that used in the previous reports (heterotrophic or mixotrophic condition). In this study, co-cultivation of microalgae obtained higher lipid productivity than mono-cultivation because of the enhancement in total lipid production and reduction in incubation time. The synergistic effects between two microalgae and nutrient-starvation conditions may be related to the increased lipids yield in co-cultivation conditions. It was reported that microalgae released more free fatty acids into media in co-cultivation conditions than that in normal culture conditions (24e26). The increased free fatty acids, key precursors in the biosynthesis of lipids, may stimulate the transcription of downstream genes involved in lipid biosynthesis (27,28). Besides, nutrient-starvation, especially nitrogen starvation was more likely to occur in co-cultivation conditions. However, nitrogen starvation may increase the intracellular content of acetylCoA, which is a major element required for the biosynthesis of
triglycerides, mainly neutral lipids (29,30). Thus, total lipid production was improved in a co-culture system. Nitrogen starvation might appear early or easily in microalgae in a co-culture system. Our data show that nitrate consumption in co-cultivation condition was significantly stronger than that in mono-cultivation conditions (Fig. 1B). It was possible that microalgae could be induced by cocultivation conditions to produce particularly high concentrations of chemical compounds such as peptides, amino acids and nitric oxide (31). Therefore, lipid accumulation of microalgae U4341 and FXY-10 were both triggered under nitrogen depleted co-cultivation condition, and the total lipid production subsequently improved. A similar conclusion was obtained from the experimental results. We calculated the dry weights of U4341 and FXY-10 to be approximately 1.03 and 0.27 (g L1) in co-cultivation, according to the proportion of cell density (Fig. 1A) and relationship of dry weight (Table 1). Lipid production values of U4341, FXY-10, and cocultivation were 467.7, 460.3 and 590.4 (mg L1), respectively. We determined which algal improves lipid production in co-cultivation. If the lipid content (32.03%) of U4341 was unchanged, then its lipid production was possibly 330 (mg L1) in mono-culture conditions. The biomass of FXY-10 (approximately 0.27 g L1) accumulated lipid production of 260.4 (mg L1). However, the lipid content of FXY-10 could be increased from 51.72% (mono-cultivation) to 96% (co-cultivation) to compensate the shortage of lipid production, which was not accomplished in the microalgae culture. The hypothesis of poison was rejected. Thus, the two microalgae contributed in improving lipid production. The culture time was shortened in co-culture condition (20 d vs. 26 d) because nitrogen consumption and the lipid accumulation were faster in co-cultivation. Therefore, biomass productivity and lipid productivity significantly increased (62 and 29.52 mg L1 d1) by shortening the culture time in co-cultivation. CO2 also greatly effects microalgae growth and lipid accumulation (32). Future investigations that will take advantage of this discovery should regulate the CO2 concentration in co-cultivation conditions to enhance lipid productivity. C. vulgaris Beijerink was first observed in algae to release substances affecting the growth of other organisms (33). Further investigations revealed that these substances were not a single compound but a mixture of fatty acids (34). The mixture was mainly composed of stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), and palmitic acid (C16:0) (24,35). Chiang et al. (35) observed the highest toxicity with linolenic acid, followed by linoleic, oleic, and palmitic acids. We obtained similar results in this experiment. The two algae grown in co-cultivation possessed significantly higher contents of oleic, linolenic, and linoleic acids than those in mono-cultivation. Thus, algal U4341 was forced in producing more C18 fatty acids, especially linolenic acid, to inhibit the growth of FXY-10 in co-cultivation under competition pressure, than that in mono-cultivation with relatively free cultivation circumstances. However, FXY-10 also generated fatty acids as competitive arms but exhibited low tolerance capacity, which resulted in growth suppression. By contrast, U4341 could strongly tolerate the toxicity against high concentration of C18 fatty acids, and growth was almost unchanged or only slightly inhibited. Not all algal oils are satisfactory for biodiesel production, but suitable oils do occur commonly. The fatty acid profiles of cocultivation in this study were mainly 16 to 18 carbon atoms, which are suitable for biodiesel production (2). The lipid quality analyzed in both species suggests that a biodiesel derived from these oils may present an acceptable cetane number (CN), iodine value (IV), and CFPP (36). The DU of the oil defined by Ramos et al. (14) is an important parameter in determining the CN and IV of the final biodiesel product. The DU for the algal oil extracted from the biomass in co-cultivation was 95.82 (Table 2), which was lower
Please cite this article in press as: Zhao, P., et al., Enhancing lipid productivity by co-cultivation of Chlorella sp. U4341 and Monoraphidium sp. FXY-10, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2013.12.014
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than those of U4341 (102.65), oils of vegetable rape (121.9), sunflower (152.2) (14), and the microalgae Ankistrodesmus fusiformis (102.91), Kirchneriella lunaris (112.77), Botryococcus braunii (100.69) (37), and Auxenochlorella protothecoides (113.5) (38). Oils that have DU higher than 137 do not meet the European biodiesel standard for CN and IV (UNE-EN 14214) (38). However, the low percentage of polyunsaturated FAME (lower DU) composes a group that is more suitable for generating biodiesel with shorter ignition time (higher CN), less deposit formation, and better lubricity (lower IV), and produces better methyl ester fuels with higher oxidation stability (37,38). Although palm oil obtained a lower DU value (64.2) (14) than co-cultivation in this study, it had the poorest result of CFPP (10 C) (Table 2). CFPP is usually used to predict the flow performance of biodiesel at low temperatures (39). The higher will be the value of CFPP, and the worse their low temperature properties (14). The EN 14214 (biodiesel standard of UK) and ASTM D6751-07a (biodiesel standard of US) both do not mention a low-temperature parameter in their list of specifications, but CSN 65 6507 (biodiesel standard of Czech) and SS 155436 (biodiesel standard of Sweden) both specify temperature limits (5 C) to the CFPP. If liquid biodiesel is cooled, palm biodiesel with high methyl esters content of stearic and palmitic acid will typically constitute a major share of material recovered from clogged biodiesel fuel filters (40). The biodiesel of microalgae in co-cultivation had better CFPP values (6.64 C), which implies good low-temperature filterability. Thus, U4341 and FXY-10 in co-cultivation could be recognized as good lipid producers for biodiesel based on the values of DU and CFPP. ACKNOWLEDGMENTS This research was supported by the National High Technology Research and Development Program of China (863 Program; no. 2008AA02Z202), the National Natural Science Foundation of China (no. 31160229) to Z. Huang and the National Natural Science Foundation of China (no. 21266013) to X. Yu. References 1. Lang, X., Dalaia, A. K., Bakhshia, N. N., Reaneyb, M. J., and Hertz, P. B.: Preparation and characterization of bio-diesels from various bio-oils, Bioresour. Technol., 80, 53e62 (2001). 2. Huang, G. H., Chen, F., Wei, D., Zhang, X. W., and Chen, G.: Biodiesel production by microalgal biotechnology, Appl. Energ., 87, 38e46 (2010). 3. Chisti, Y.: Biodiesel from microalgae, Biotechnol. Adv., 25, 294e306 (2007). 4. Alptekin, E. and Canakci, M.: Optimization of pretreatment reaction for methyl ester production from chicken fat, Fuel, 89, 4035e4039 (2010). 5. Oncel, S. S., Imamoglu, E., Gunerken, E., and Sukan, F. V.: Comparison of different cultivation modes and light intensities using mono-cultivations and co-cultivations of Haematococcus pluvialis and Chlorella zofingiensis, J. Chem. Technol. Biot., 86, 414e420 (2011). 6. Shu, C. H., Tsai, C. C., Chen, K. Y., Liao, W. H., and Huang, H. C.: Enhancing high quality oil accumulation and carbon dioxide fixation by a mixed culture of Chlorella sp. and Saccharomyces cerevisiae, J. Taiwan Inst. Chem. Eng., 44, 936e942 (2013). 7. Bai, R. and Gutierrez, M. T.: Effect of organic carbon, C:N ratio and light on the growth and lipid productivity of microalgae/cyanobacteria co-culture, Eng. Life Sci., 14, 47e56 (2014). 8. Liang, Y., Sarkany, N., and Cui, Y.: Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions, Biotechnol. Lett., 31, 1043e1049 (2009). 9. Bogen, C., Klassen, V., Wichmann, J., Russa, M. L., Doebbe, A., Grundmann, M., Uronen, P., Kruse, O., and Mussgnug, J. H.: Identification of Monoraphidium contortum as a promising species for liquid biofuel production, Bioresour. Technol., 133, 622e626 (2013). 10. Yu, X., Zhao, P., He, C., Li, J. J., Tang, X. H., Zhou, J. P., and Huang, Z. X.: Isolation of a novel strain of Monoraphidium sp. and characterization of its potential application as biodiesel feedstock, Bioresour. Technol., 121, 256e262 (2012). 11. Hecht, U. and Mohr, H.: Factors controlling nitrate and ammonium accumulation in mustard (Sinapis alba) seedlings, Physiol. Plant., 78, 379e387 (1990).
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Please cite this article in press as: Zhao, P., et al., Enhancing lipid productivity by co-cultivation of Chlorella sp. U4341 and Monoraphidium sp. FXY-10, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2013.12.014