Bioprocess Biosyst Eng DOI 10.1007/s00449-014-1238-x
ORIGINAL PAPER
Growth and biochemical composition of filamentous microalgae Tribonema sp. as potential biofuel feedstock Hui Wang • Bei Ji • Junfeng Wang • Fajin Guo • Wenjun Zhou • Lili Gao Tian Zhong Liu
•
Received: 21 March 2014 / Accepted: 9 June 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Filamentous oleaginous microalgae Tribonema minus have advantages in relatively easy harvesting and grazers resistance in mass cultivation due to its filaments in previous study. To evaluate whether the genus Tribonema is a valuable candidate for use in biofuel production, the morphology, growth, biochemical composition and fatty acid profile of six filamentous microalgae strains Tribonema sp. were investigated. All the strains are unbranched filament in single row of elongated cylinder, attaining 0.5–3 mm in length. The growth rates of tested strains were 0.35–0.42 g L-1 d-1. Generally, for all strains, decrease in protein content was followed by a slight increase in lipid and significant increase in carbohydrate in early phase, afterwards, lipid increased constantly inversely to decrease in carbohydrate content. After 15-day cultivation, total lipid contents of tested strains ranged from 38–61 %, of which TAG were the majority and palmitic acid (C16:0) and palmitoleic acid (C16:1) were the dominant
W. Hui and J. Bei contributed equally to this work. H. Wang B. Ji J. Wang F. Guo W. Zhou L. Gao T. Z. Liu (&) Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, People’s Republic of China e-mail: [email protected] B. Ji F. Guo University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China B. Ji College of Chemical and Environmental Engineering, Shandong University of Science and Technology, Qingdao, Shandong 266590, People’s Republic of China
components. The study confirmed that the genus Tribonema is the potential for biodiesel and bioethanol production upon culture time. Keywords Tribonema sp. Morphology Biochemical composition Lipid Fatty acid
Introduction Limited fossil fuel reserves of the earth have been rapidly depleted and there is an increasing need for renewable energy sources, especially biofuels [1]. Microalgae, which grow fast in variety of water environments to accumulate lipids and polysaccharides, exhibit advantages to be the potential source for biodiesel and/or bioethanol production when compared with terrestrial plants [2, 3]. Many researches have been tried to set up the microalgae biofuel process, however, no commercial system has succeeded in economic viability, mainly due to the technical and cost problems related with the microalgae biomass production [4, 5]. Microalgae strains and mass cultivation are thought to be the core technologies of microalgae biofuel industrialization. Besides the biological behaviors of fast growth and high lipid content, more industrial properties including robustness to contamination, ease to harvest, etc., for oleaginous microalgae specie should be more emphasized to ensure the success of mass cultivation and low cost [6]. To date, studies on microalgae are mainly concentrated in the unicellular microalgae with the size \30 lm [7, 8]. However, in the outdoor scale culture, such nutrient-rich algal cells with tiny size are usually palatable for grazers (ciliate, amoeba and rotifera) to promote their massive proliferation during cultivation and as a result, the mass cultivation of microalgae usually crashed down. In
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addition, the harvesting of unicellular microalgae with tiny size from culture medium is another tough work which related with the power consumption and biomass quality and cost [9]. Centrifuging is the usual way to harvest the unicellular microalgae, but it is time consuming and costly [10]. Filtration, gravitational sedimentation and dissolvedair flotation are the other options to harvest microalgae from medium; however, their harvesting efficiency is usually dominated by the formation of floccule of tiny unicellular algae cells induced by flocculent additives. The flocculant residuals in both algal biomass and harvested water are not only negative for later processing, but also disadvantages for culture medium recycling [11]. Kim et al. [12] reported that filamentous microalgae Spirulina platensis could be harvested economically by filtration or flotation without any flocculants. Moreover, Wang et al. [13] introduced that microalgae cells with bigger size might have grazer resistance. It is reasonable to believe that those filamentous species with large size could overcome above puzzling problems. Therefore, selection of filamentous microalgae with high lipid content is the key to the future success of algal biofuels [6]. Fortunately, a filamentous microalgal strain Tribonema minus was proved as a potential candidate for bidodiesl production in our preliminary evaluation, due to fast growth rate, high lipid content, bigger size, grazers resistance and relatively easy harvesting [14]. And an integrated process of biodiesel production from Tribonema minus was successfully built up. It is known that the filamentous microalgal genus Tribonema are common in freshwater environments worldwide [15], and the group includes about 100 genera. Whether other Tribonema genera have the potential to be the biofuel algal strains? Here, six Tribonema sp. strains were investigated from their morphologies, biomass and biochemical compositions. It is expected to provide more options of microalgae species and evaluate the feasibility of Tribonema genus as the feedstock for biofuel production.
Materials and methods
light and bubbled with compressed air containing 1.5 % CO2 (v/v). Nitrogen utilization in tested microalgal strain culture For nitrogen analyses, 10 mL of culture were filtered (0.2 lm), the filtrate were analyzed for concentrations using nutrient autoanalyser (Vario EL cube, Elementary Inc., Germany). Description of cell sizes and morphologies A 20 lL sample was taken from each column of strain and placed in a slide, and then covered with cover glass. The slide was placed under microscope and the cellular morphologies (BX51, Olympus, Germany), especially the sizes of tested strains were detected. Each sample was analyzed in triplicate. Determination of biomass and biochemical composition A certain volume (V) of algal culture was filtered onto a preweighed 0.45 lm GF/C filter membrane (Whatman, W0). The membrane was oven dried at 105 °C overnight and then weighted (W1). The biomass density of culture was calculated as (W1 - W0)/V. The total nitrogen content of the microalgae was first detected by an elemental analyzer, and then the crude protein concentration was obtained according to the correlation reported in the literature (i.e., protein concentration = nitrogen content 9 6.25). The carbohydrate content and composition in microalgae were determined using the modified quantitative saccharification (QS) method reported by the National Renewable Energy Laboratory (NREL), USA [16]. The total lipid content was determined by gravimetric analysis according to modified Bligh and Dyer’s method [17]. Approximately 50 mg of dried algal pellet was ground with quart firstly and then mixed with 7.5 ml chloroform/methanol (1:2, v/v) at 37 °C overnight and then centrifuged. The supernatant was collected and residual biomass was extracted twice. The supernatants were combined, and chloroform and 1 % sodium chloride solution were added to a final volume ratio of 1:1:0.9 (chloroform:methanol:water).
Algae and culture conditions Analysis of lipid composition and fatty acid profiles The filamentous microalgae T. aequale 200.80, T. aequale 880-1, T. minus 880-3, T. ulotrichoides 21.94, T. utriculosum 22.94, T. vulgare 24.94 used in this study was purchased from the Culture Collection of Algae of Gottingen University. Column photo bioreactor (40 cm height, 2 cm width) with 200 mL of medium settled as suspended cultures to cultivate these filamentous microalgae. Cultures were grown under 100 lmol photons m-2 s-1 of artificial
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Lipid composition was analyzed using a thin-layer chromatography (TLC) system (TLC-FID, MK-6, Iatron Laboratories, Inc., Japan) [18]. Samples were dissolved in chloroform to a concentration of 5–8 mg ml-1 and spotted onto Chromarod S-III silica coated quartz rods held in a frame. The rods were developed in a solvent system of benzene:chloroform:methanol (150:60:2, v/v/v) for the first
Bioprocess Biosyst Eng
Fig. 1 Cellular morphologies of tested strains. a T. vulgare 24.94, b T. aequale 200.80, c T. aequale 880-1, d T. ulotrichoides 21.94, e T. utriculosum 22.94, f T. minus 880-3
migration to 7 cm, followed by a solution of benzene:hexane (50:50, v/v) for the second migration to 10 cm. The individual lipid components were identified by co-chromatography with pure standards (SE; FAME; FFA; TAG; DAG; MAG; PL, purchased from Sigma, St. Louis, MO, USA). The quantity of TAG was estimated from the peak areas of pure standards. Fatty acid profiles in microalgae cells were determined post-conversion to fatty acid methyl esters (FAMEs) [19]. And then a 0.5-mg sample was dissolved in 1 mL heptane containing 50 lg nonadecanoic acid as internal standard for FAME analysis on a Varian 450GC (Varian Inc., USA) equipped with a flame ionization detector (FID) and Agilent HP-5 GC Capillary Column (30 m 9 0.25 mm 9 0.25 lm). The individual FAMEs were identified by chromatographic comparison with authentic standards (Sigma). The quantities of individual FAMEs were calculated as W = ms 9 fi 9 Ai/ (m 9 As) 9 100 %, where W is the relative content of each fatty acid, presented in a percentage of total fatty acid; ms is the mass of internal standard, fi is the coefficient value of section i, Ai is the peak area of section i, m is the weight of sample and As is the area of standard.
24.94 are with 7–10 lm wide and 16–21 lm long while the cells of T. aequale 200.80 are with 4–7 lm wide and 8–17 lm long (Fig. 1a, b). The width and length of T. ulotrichoides 21.94 cells are 6–10 and 10–20 lm, which is slight bigger than T. aequale 880-1 which is with 3–5 lm wide and 7–13 lm long (Fig. 1c, d). The cells of T. utriculosum 22.94 are with 9–15 lm wide and 17–29 lm long, and that of T. minus 880-3 are with 6–8 lm wide and 13–16 lm long. Data above revealed that even single cell of Tribonema sp. (3–15 lm wide and 7–20 lm long) is bigger than common unicellular microalgae, such as Chlorella sp. (3–8 lm) and Nannochloropsis sp. (2–4 lm). Moreover, Tribonema is relatively large, attaining sizes of 0.5–3 mm filament in length, which is longer than the size of main grazers, such as ciliate (5–200 lm) and rotifer (0.01–0.5 mm). The longer size perhaps makes the strains unpalatable to grazers to contribute to their pest resistance. In a previous study, cells of Tribonema minus were successfully harvested by dissolved-air flotation (DAF) without any flocculants [14]. The similar size of tested strains indicated a good flotation activity of filamentous microalgae Tribonema sp.
Results
Growth of algae in column photobioreactor
Cellular morphologies of tested strains
Growth curve of tested microalgae in terms of biomass was presented in Fig. 2. The tested microalgal strains revealed the similar biomass concentration during 15 days. From inoculation concentration of ca. 0.093 g L-1, high biomass (g L-1) of 5.792, 6.422, 5.319, 5.248, 5.543, 5.853 were
As shown in Fig. 1, all tested strains have unbranched filament composed of a single row of elongated, cylindrical cells, as described by Ivan [20]. The cells of T. vulgare
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Fig. 2 Growth curve of tested microalgal strains
found for T. vulgare 24.94, T. aequale 200.80, T. aequale 880-1, T. ulotrichoides 21.94, T. utriculosum 22.94, T. minus 880-3, respectively. Most microalgal strains had biomass productivity, i.e., 0.344–0.380 g L-1 d-1, except T. aequale 200.80 had higher biomass productivity of 0.422 g L-1 d-1. In addition, most strains had a growth cycle of 12 days to stationary phase. The biomass productivities of tested microalgae strains were ca. 0.35–0.42 g L-1 d-1, which was lower than those of unicellular microalgae such as Chlorella sp. (0.44– 0.62 g L-1 d-1), Scenedesmus sp. (0.59–0.95 g L-1 d-1), Nannochloropsis sp. (0.42–0.55 g L-1 d-1) in similar culture scale and light condition [21, 22]. Light supply the energy makes it is an important parameter in the growth and lipid accumulation of microalgae [23]. During the cultivation, cells of unicellular microalgae were more easily mixed than filamentous microalgae, resulting in the different uniformities of light absorption, which means although external energy supply is equal, the rates of energy absorption and utilization between single-cell microalgae and filamentous microalgae are different. Therefore, improving the external energy supply might be an effective way to speed up the growth rate of filamentous microalgae. Changes in protein, total lipid and carbohydrate The biochemical compositions of tested microalgal strains were measured by day 7 and day 14, respectively (Fig. 3). Total protein in the algal samples decreased in different degrees in response to the culture time. From initial contents of ca. 22.3–30.9 % of dry weight (Fig. 3a), 30.1, 42.1, 53.6, 39.4, 55.4 and 44.4 % decreases in protein contents were observed after 7-day culture (Fig. 3b) and decreases of 38.6, 37.3, 54.2, 58.1, 70.3 and 63.2 % were observed by day 14 (Fig. 3c) in T. vulgare 24.94, T. aequale 200.80, T. aequale 880-1, T. ulotrichoides 21.94, T. utriculosum 22.94 and T. minus 880-3, respectively.
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Fig. 3 Biochemical composition contents of tested microalgae. a day 0, b day 7, c day 14
Corresponding methods were adopted to detect the contents and compositions of carbohydrate in tested microalgal strains. No starch was found in any tested algae, because the cytoplasm of genus Tribonema contains no pyrenoid [24]. The carbohydrates of Tribonema include small amount of soluble sugar and large amount of insoluble polysaccharides because the cell wall of genus Tribonema filament is mainly made up of cellulose. Thus, the carbohydrate content shown in Fig. 3 was the sum of the contents of soluble sugar and cellulose. As shown in Fig. 3, there were slight increases in carbohydrate in most tested strains by day 7 (Fig. 3b) from an initial value of 40.45–53.23 % of dry weight (Fig. 3a), subsequently, the carbohydrate contents declined linearly to ca. 25.52–39.07 % of dry weight inversely to the increase in lipid content by day 14 (Fig. 3c). Based on methanol–chloroform extract and gravimetric method, the initial total lipid contents were ca.
Bioprocess Biosyst Eng Table 1 N–NO3- concentration in the medium after 15-day cultivation of six strains Strains
T.v 24.94
T.a 200.80
T.a 880-1
T.u 21.94
T.u 22.94
T.m 880-3
N–NO3- concentration (mg/L)
126.06 ± 7.5
174.82 ± 4.8
155.61 ± 5.2
162.5 ± 6.7
148.43 ± 2.9
169.7 ± 7.3
16.5–20.5 % of dry weight (Fig. 3a). There were no obvious increases in lipid contents of dry weight in T. vulgare 24.94, T. aequale 200.80, T. aequale 880-1 and T. ulotrichoides 21.94 by day 7 (Fig. 3b); however, the lipid contents of dry weight in T. utriculosum 22.94 and T. minus 880-3 increased to 26.1 and 35.6 % from 18.6 and 20.5 %, respectively. After 14-day cultivation, the lipid contents of dry weight in T. vulgare 24.94, T. aequale 200.80, T. aequale 880-1, T. ulotrichoides 21.94, T. utriculosum 22.94 and T. minus 880-3 achieved to 53.2, 48.8, 38.9, 46.8, 52.9 and 61.8 %, respectively (Fig. 3c). In general, decrease in protein content of dry weight was followed by a simultaneous increase in carbohydrate and lipid contents in all tested strains during the early phase of cultivation. Moreover, carbohydrate content increment was faster and higher than increase in lipid content of dry weight. In the later phase of cultivation, the protein content of dry weight decreased constantly, meanwhile, lipid content increased dramatically while carbohydrate decreased by day 14. This may indicate that Tribonema sp. first accumulate carbohydrate and then induced lipid synthesis, similar condition also appeared in Chlamydomonas reinhardtii reported by [25]. The difference was that C. reinhardtii first accumulated starch and then lipid under nitrogen starvation. However, nitrogen was always sufficient during 15-day cultivation in this study (Table 1). Why the carbohydrate decreased while lipid increased in later phase of cultivation under nitrogen-replete condition will be observed in further study. On the other hand, potential competition between synthesis of lipid and carbohydrates is an important factor when deciding optimal biofuel production strategies. Microalgae lipid is used for biodiesel production while carbohydrate are excellent substrate for bioethanol production [26]. Tribonema sp. based carbohydrates are mainly in the form of cellulose (with the absence of lignin) are thus much easier to convert to monosaccharides when compared with lignocellulosic materials [27]. Therefore, Tribonema sp. biomass contains abundant carbohydrate could be considered for bioethanol fermentation, while biomass contains lipid for biodiesel production. Lipid composition and fatty acid profiles Triacylglycerols (TAG) are stored in specialized cytosolic oil bodies and function as energy reserve. Therefore, quantities and fatty acid compositions of triacylglycerols (TAG) in tested stains are important factors to evaluate
Fig. 4 Lipid compositions of tested microalgal strains cultured for 14 days
whether they can serve as feedstock for biodiesel production [28]. Lipid compositions of tested microalgae strains cultured by day 14 were analyzed using TLC-FID (Fig. 4). Sterol esters (SE), triacylglycerols (TAG), digalactosyl diacyglycerol (DAG), and phospholipid (PL) were presented in tested strains. TAGs accounted for 66.2, 78.9, 71.1, 75.1, 81.3 and 81.4 % of dry weights in T. vulgare 24.94, T. aequale 200.80, T. aequale 880-1, T. ulotrichoides 21.94, T. utriculosum 22.94 and T. minus 880-3, respectively. Obviously, TAG was the majority ingredient in total lipid in all tested strains under normal conditions, revealing the specie of Tribonema could be a potential raw material for biodiesel production. Phospholipids were the second ingredient in total lipids from tested strains, 15.1, 5.7, 22.2, 18.4, 15.4 and 12.5 % of phospholipids were detected in T. vulgare 24.94, T. aequale 200.80, T. aequale 880-1, T. ulotrichoides 21.94, T. utriculosum 22.94, T. minus 880-3, respectively. The lowest ratio of TAG/PL approached to 3.2 presented in T. aequale 880-1 yet the highest one accounted for 6.52 in T. minus 880-3. Such higher TAG/PL lipid predicts a relatively easier and better biodiesel conversion. Fatty acids in tested strains were primarily esterified and the major fatty acid composition of each was determined using GC–MS analysis (Table 2). The major fatty acids in the six strains were myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1), oleic acid (C18:1) comprising 6.9–12.1 %, 19.25–33.68 %, 38.49–51.21 % and 2.96–7.28 % of the total fatty acids, respectively, whereas myristoleic (C14:1), pentadecanoic (C15:0), hexadecadioneic acid (C16:2) and linoleic acid (C18:2) existed as minor fatty acids. Table 2 also revealed that saturated
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Bioprocess Biosyst Eng Table 2 Fatty acid composition of tested microalgal strains (% of total FAME) Properties
Strains T.v 24.94
T.a 200.80
T.a 880-1
T.u 21.94
T.u 22.94
T.m 880-3
C14:0
10.73
9.31
9.09
12.03
11.97
6.85
C14:1
0.62
nd
nd
0.54
0.98
nd
C15:0
0.46
nd
nd
0.86
1.07
0.56
C16:0
26.32
24.53
27.65
33.68
19.25
28.35
C16:1
42.5
51.1
45.93
38.49
51.21
50.65
C16:1
nd
nd
0.50
nd
nd
nd
C16:2
nd
0.72
0.58
0.68
nd
0.67
C16:2
nd
nd
nd
0.79
0.01
0.88
C18:0
1.72
0.66
1.06
1.2
0.99
1.02
C18:1
7.28
4.71
4.54
3.12
6.92
2.96
C18:2
0.63
0.55
0.65
nd
0.54
0.71
C20:3
1.82
1.56
1.75
1.43
1.74
1.20
C20:4
2.53
1.34
2.11
3.02
3.17
3.02
C20:5 SFA
5.32 39.23
5.45 35.56
6.12 37.81
4.15 46.78
2.15 33.29
3.14 36.78
MUFA
50.38
54.19
50.47
42.15
57.11
52.51
PUFA
10.3
9.62
11.21
10.7
7.61 ± 1.3
9.61
and monounsaturated fatty acids were the dominant components, comprising 88.28–90.4 % of the total fatty acids in the biodiesels from the tested algae. In addition, eicosapentaenoic acid (C20:5) was the main component of polyunsaturated fatty acids in all tested strains. Biodiesel reduces emissions of CO2, CO, hydrocarbons and particle emissions; however, such biodiesel also suffers from several performance-related problems including poor cold flow properties and insufficient oxidative stability [29]. The most common fatty acid methyl esters present in biodiesel are palmitic acid (16:0), steric acid (18:0), oleic acid (18:1), linoleic acid (18:2) and linolenic acid (18:3) [30]. However, palmitoleic acid (16:1) instead of linolenic acid (18:3) presented in the fatty acid profiles from six Tribonema strains. The monosaturated fatty acid was the dominant component, comprising 43.15–54.19 % of the total fatty acids in lipid from six strains, which was considered to be better than polyunsaturated fatty methyl esters for improving CN and oxidative stability without any concomitant adverse effect on the cold properties of the diesel [31].
Conclusion In short, all strains of genus Tribonema are unbranched filament composed of a single row of elongated, cylindrical cells, attaining 0.5–3 mm in length. The biomass productivities of all tested Tribonema sp. strains in bubbled
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column are ca. 0.35–0.42 g L-1 d-1, similar to those of unicellular microalgae in same condition. The genus Tribonema mainly accumulates carbohydrate in early phase and then induced lipid synthesis in later phase of cultivation under nitrogen repletion. The highest total lipid contents of tested strains ranged from 38 to 61 %, of which TAG was the majority ingredient. Besides the dominant components of palmitic acid (C16:0) and palmitoleic acid (C16:1), eicosapentaenoic acid (C20:5) was also found in all tested strains. Analyses of the present results suggest that genus Tribonema is promising feedstock for bioethanol and biodiesel production and such filamentous microalgae should be paid more attention. Acknowledgments This work was supported by the Solar Energy Initiative Plan (KGCX2-EW-309) of Chinese Academy of Sciences, Director Innovation Foundation of Qingdao Institute of Bioenergy and Bioprocess Technology, CAS (Y37204110E) and the Science and Technology Development Planning of Shandong Province (2013GGF01008).
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ORIGINAL PAPER
Growth and biochemical composition of filamentous microalgae Tribonema sp. as potential biofuel feedstock Hui Wang • Bei Ji • Junfeng Wang • Fajin Guo • Wenjun Zhou • Lili Gao Tian Zhong Liu
•
Received: 21 March 2014 / Accepted: 9 June 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Filamentous oleaginous microalgae Tribonema minus have advantages in relatively easy harvesting and grazers resistance in mass cultivation due to its filaments in previous study. To evaluate whether the genus Tribonema is a valuable candidate for use in biofuel production, the morphology, growth, biochemical composition and fatty acid profile of six filamentous microalgae strains Tribonema sp. were investigated. All the strains are unbranched filament in single row of elongated cylinder, attaining 0.5–3 mm in length. The growth rates of tested strains were 0.35–0.42 g L-1 d-1. Generally, for all strains, decrease in protein content was followed by a slight increase in lipid and significant increase in carbohydrate in early phase, afterwards, lipid increased constantly inversely to decrease in carbohydrate content. After 15-day cultivation, total lipid contents of tested strains ranged from 38–61 %, of which TAG were the majority and palmitic acid (C16:0) and palmitoleic acid (C16:1) were the dominant
W. Hui and J. Bei contributed equally to this work. H. Wang B. Ji J. Wang F. Guo W. Zhou L. Gao T. Z. Liu (&) Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, People’s Republic of China e-mail: [email protected] B. Ji F. Guo University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China B. Ji College of Chemical and Environmental Engineering, Shandong University of Science and Technology, Qingdao, Shandong 266590, People’s Republic of China
components. The study confirmed that the genus Tribonema is the potential for biodiesel and bioethanol production upon culture time. Keywords Tribonema sp. Morphology Biochemical composition Lipid Fatty acid
Introduction Limited fossil fuel reserves of the earth have been rapidly depleted and there is an increasing need for renewable energy sources, especially biofuels [1]. Microalgae, which grow fast in variety of water environments to accumulate lipids and polysaccharides, exhibit advantages to be the potential source for biodiesel and/or bioethanol production when compared with terrestrial plants [2, 3]. Many researches have been tried to set up the microalgae biofuel process, however, no commercial system has succeeded in economic viability, mainly due to the technical and cost problems related with the microalgae biomass production [4, 5]. Microalgae strains and mass cultivation are thought to be the core technologies of microalgae biofuel industrialization. Besides the biological behaviors of fast growth and high lipid content, more industrial properties including robustness to contamination, ease to harvest, etc., for oleaginous microalgae specie should be more emphasized to ensure the success of mass cultivation and low cost [6]. To date, studies on microalgae are mainly concentrated in the unicellular microalgae with the size \30 lm [7, 8]. However, in the outdoor scale culture, such nutrient-rich algal cells with tiny size are usually palatable for grazers (ciliate, amoeba and rotifera) to promote their massive proliferation during cultivation and as a result, the mass cultivation of microalgae usually crashed down. In
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addition, the harvesting of unicellular microalgae with tiny size from culture medium is another tough work which related with the power consumption and biomass quality and cost [9]. Centrifuging is the usual way to harvest the unicellular microalgae, but it is time consuming and costly [10]. Filtration, gravitational sedimentation and dissolvedair flotation are the other options to harvest microalgae from medium; however, their harvesting efficiency is usually dominated by the formation of floccule of tiny unicellular algae cells induced by flocculent additives. The flocculant residuals in both algal biomass and harvested water are not only negative for later processing, but also disadvantages for culture medium recycling [11]. Kim et al. [12] reported that filamentous microalgae Spirulina platensis could be harvested economically by filtration or flotation without any flocculants. Moreover, Wang et al. [13] introduced that microalgae cells with bigger size might have grazer resistance. It is reasonable to believe that those filamentous species with large size could overcome above puzzling problems. Therefore, selection of filamentous microalgae with high lipid content is the key to the future success of algal biofuels [6]. Fortunately, a filamentous microalgal strain Tribonema minus was proved as a potential candidate for bidodiesl production in our preliminary evaluation, due to fast growth rate, high lipid content, bigger size, grazers resistance and relatively easy harvesting [14]. And an integrated process of biodiesel production from Tribonema minus was successfully built up. It is known that the filamentous microalgal genus Tribonema are common in freshwater environments worldwide [15], and the group includes about 100 genera. Whether other Tribonema genera have the potential to be the biofuel algal strains? Here, six Tribonema sp. strains were investigated from their morphologies, biomass and biochemical compositions. It is expected to provide more options of microalgae species and evaluate the feasibility of Tribonema genus as the feedstock for biofuel production.
Materials and methods
light and bubbled with compressed air containing 1.5 % CO2 (v/v). Nitrogen utilization in tested microalgal strain culture For nitrogen analyses, 10 mL of culture were filtered (0.2 lm), the filtrate were analyzed for concentrations using nutrient autoanalyser (Vario EL cube, Elementary Inc., Germany). Description of cell sizes and morphologies A 20 lL sample was taken from each column of strain and placed in a slide, and then covered with cover glass. The slide was placed under microscope and the cellular morphologies (BX51, Olympus, Germany), especially the sizes of tested strains were detected. Each sample was analyzed in triplicate. Determination of biomass and biochemical composition A certain volume (V) of algal culture was filtered onto a preweighed 0.45 lm GF/C filter membrane (Whatman, W0). The membrane was oven dried at 105 °C overnight and then weighted (W1). The biomass density of culture was calculated as (W1 - W0)/V. The total nitrogen content of the microalgae was first detected by an elemental analyzer, and then the crude protein concentration was obtained according to the correlation reported in the literature (i.e., protein concentration = nitrogen content 9 6.25). The carbohydrate content and composition in microalgae were determined using the modified quantitative saccharification (QS) method reported by the National Renewable Energy Laboratory (NREL), USA [16]. The total lipid content was determined by gravimetric analysis according to modified Bligh and Dyer’s method [17]. Approximately 50 mg of dried algal pellet was ground with quart firstly and then mixed with 7.5 ml chloroform/methanol (1:2, v/v) at 37 °C overnight and then centrifuged. The supernatant was collected and residual biomass was extracted twice. The supernatants were combined, and chloroform and 1 % sodium chloride solution were added to a final volume ratio of 1:1:0.9 (chloroform:methanol:water).
Algae and culture conditions Analysis of lipid composition and fatty acid profiles The filamentous microalgae T. aequale 200.80, T. aequale 880-1, T. minus 880-3, T. ulotrichoides 21.94, T. utriculosum 22.94, T. vulgare 24.94 used in this study was purchased from the Culture Collection of Algae of Gottingen University. Column photo bioreactor (40 cm height, 2 cm width) with 200 mL of medium settled as suspended cultures to cultivate these filamentous microalgae. Cultures were grown under 100 lmol photons m-2 s-1 of artificial
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Lipid composition was analyzed using a thin-layer chromatography (TLC) system (TLC-FID, MK-6, Iatron Laboratories, Inc., Japan) [18]. Samples were dissolved in chloroform to a concentration of 5–8 mg ml-1 and spotted onto Chromarod S-III silica coated quartz rods held in a frame. The rods were developed in a solvent system of benzene:chloroform:methanol (150:60:2, v/v/v) for the first
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Fig. 1 Cellular morphologies of tested strains. a T. vulgare 24.94, b T. aequale 200.80, c T. aequale 880-1, d T. ulotrichoides 21.94, e T. utriculosum 22.94, f T. minus 880-3
migration to 7 cm, followed by a solution of benzene:hexane (50:50, v/v) for the second migration to 10 cm. The individual lipid components were identified by co-chromatography with pure standards (SE; FAME; FFA; TAG; DAG; MAG; PL, purchased from Sigma, St. Louis, MO, USA). The quantity of TAG was estimated from the peak areas of pure standards. Fatty acid profiles in microalgae cells were determined post-conversion to fatty acid methyl esters (FAMEs) [19]. And then a 0.5-mg sample was dissolved in 1 mL heptane containing 50 lg nonadecanoic acid as internal standard for FAME analysis on a Varian 450GC (Varian Inc., USA) equipped with a flame ionization detector (FID) and Agilent HP-5 GC Capillary Column (30 m 9 0.25 mm 9 0.25 lm). The individual FAMEs were identified by chromatographic comparison with authentic standards (Sigma). The quantities of individual FAMEs were calculated as W = ms 9 fi 9 Ai/ (m 9 As) 9 100 %, where W is the relative content of each fatty acid, presented in a percentage of total fatty acid; ms is the mass of internal standard, fi is the coefficient value of section i, Ai is the peak area of section i, m is the weight of sample and As is the area of standard.
24.94 are with 7–10 lm wide and 16–21 lm long while the cells of T. aequale 200.80 are with 4–7 lm wide and 8–17 lm long (Fig. 1a, b). The width and length of T. ulotrichoides 21.94 cells are 6–10 and 10–20 lm, which is slight bigger than T. aequale 880-1 which is with 3–5 lm wide and 7–13 lm long (Fig. 1c, d). The cells of T. utriculosum 22.94 are with 9–15 lm wide and 17–29 lm long, and that of T. minus 880-3 are with 6–8 lm wide and 13–16 lm long. Data above revealed that even single cell of Tribonema sp. (3–15 lm wide and 7–20 lm long) is bigger than common unicellular microalgae, such as Chlorella sp. (3–8 lm) and Nannochloropsis sp. (2–4 lm). Moreover, Tribonema is relatively large, attaining sizes of 0.5–3 mm filament in length, which is longer than the size of main grazers, such as ciliate (5–200 lm) and rotifer (0.01–0.5 mm). The longer size perhaps makes the strains unpalatable to grazers to contribute to their pest resistance. In a previous study, cells of Tribonema minus were successfully harvested by dissolved-air flotation (DAF) without any flocculants [14]. The similar size of tested strains indicated a good flotation activity of filamentous microalgae Tribonema sp.
Results
Growth of algae in column photobioreactor
Cellular morphologies of tested strains
Growth curve of tested microalgae in terms of biomass was presented in Fig. 2. The tested microalgal strains revealed the similar biomass concentration during 15 days. From inoculation concentration of ca. 0.093 g L-1, high biomass (g L-1) of 5.792, 6.422, 5.319, 5.248, 5.543, 5.853 were
As shown in Fig. 1, all tested strains have unbranched filament composed of a single row of elongated, cylindrical cells, as described by Ivan [20]. The cells of T. vulgare
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Fig. 2 Growth curve of tested microalgal strains
found for T. vulgare 24.94, T. aequale 200.80, T. aequale 880-1, T. ulotrichoides 21.94, T. utriculosum 22.94, T. minus 880-3, respectively. Most microalgal strains had biomass productivity, i.e., 0.344–0.380 g L-1 d-1, except T. aequale 200.80 had higher biomass productivity of 0.422 g L-1 d-1. In addition, most strains had a growth cycle of 12 days to stationary phase. The biomass productivities of tested microalgae strains were ca. 0.35–0.42 g L-1 d-1, which was lower than those of unicellular microalgae such as Chlorella sp. (0.44– 0.62 g L-1 d-1), Scenedesmus sp. (0.59–0.95 g L-1 d-1), Nannochloropsis sp. (0.42–0.55 g L-1 d-1) in similar culture scale and light condition [21, 22]. Light supply the energy makes it is an important parameter in the growth and lipid accumulation of microalgae [23]. During the cultivation, cells of unicellular microalgae were more easily mixed than filamentous microalgae, resulting in the different uniformities of light absorption, which means although external energy supply is equal, the rates of energy absorption and utilization between single-cell microalgae and filamentous microalgae are different. Therefore, improving the external energy supply might be an effective way to speed up the growth rate of filamentous microalgae. Changes in protein, total lipid and carbohydrate The biochemical compositions of tested microalgal strains were measured by day 7 and day 14, respectively (Fig. 3). Total protein in the algal samples decreased in different degrees in response to the culture time. From initial contents of ca. 22.3–30.9 % of dry weight (Fig. 3a), 30.1, 42.1, 53.6, 39.4, 55.4 and 44.4 % decreases in protein contents were observed after 7-day culture (Fig. 3b) and decreases of 38.6, 37.3, 54.2, 58.1, 70.3 and 63.2 % were observed by day 14 (Fig. 3c) in T. vulgare 24.94, T. aequale 200.80, T. aequale 880-1, T. ulotrichoides 21.94, T. utriculosum 22.94 and T. minus 880-3, respectively.
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Fig. 3 Biochemical composition contents of tested microalgae. a day 0, b day 7, c day 14
Corresponding methods were adopted to detect the contents and compositions of carbohydrate in tested microalgal strains. No starch was found in any tested algae, because the cytoplasm of genus Tribonema contains no pyrenoid [24]. The carbohydrates of Tribonema include small amount of soluble sugar and large amount of insoluble polysaccharides because the cell wall of genus Tribonema filament is mainly made up of cellulose. Thus, the carbohydrate content shown in Fig. 3 was the sum of the contents of soluble sugar and cellulose. As shown in Fig. 3, there were slight increases in carbohydrate in most tested strains by day 7 (Fig. 3b) from an initial value of 40.45–53.23 % of dry weight (Fig. 3a), subsequently, the carbohydrate contents declined linearly to ca. 25.52–39.07 % of dry weight inversely to the increase in lipid content by day 14 (Fig. 3c). Based on methanol–chloroform extract and gravimetric method, the initial total lipid contents were ca.
Bioprocess Biosyst Eng Table 1 N–NO3- concentration in the medium after 15-day cultivation of six strains Strains
T.v 24.94
T.a 200.80
T.a 880-1
T.u 21.94
T.u 22.94
T.m 880-3
N–NO3- concentration (mg/L)
126.06 ± 7.5
174.82 ± 4.8
155.61 ± 5.2
162.5 ± 6.7
148.43 ± 2.9
169.7 ± 7.3
16.5–20.5 % of dry weight (Fig. 3a). There were no obvious increases in lipid contents of dry weight in T. vulgare 24.94, T. aequale 200.80, T. aequale 880-1 and T. ulotrichoides 21.94 by day 7 (Fig. 3b); however, the lipid contents of dry weight in T. utriculosum 22.94 and T. minus 880-3 increased to 26.1 and 35.6 % from 18.6 and 20.5 %, respectively. After 14-day cultivation, the lipid contents of dry weight in T. vulgare 24.94, T. aequale 200.80, T. aequale 880-1, T. ulotrichoides 21.94, T. utriculosum 22.94 and T. minus 880-3 achieved to 53.2, 48.8, 38.9, 46.8, 52.9 and 61.8 %, respectively (Fig. 3c). In general, decrease in protein content of dry weight was followed by a simultaneous increase in carbohydrate and lipid contents in all tested strains during the early phase of cultivation. Moreover, carbohydrate content increment was faster and higher than increase in lipid content of dry weight. In the later phase of cultivation, the protein content of dry weight decreased constantly, meanwhile, lipid content increased dramatically while carbohydrate decreased by day 14. This may indicate that Tribonema sp. first accumulate carbohydrate and then induced lipid synthesis, similar condition also appeared in Chlamydomonas reinhardtii reported by [25]. The difference was that C. reinhardtii first accumulated starch and then lipid under nitrogen starvation. However, nitrogen was always sufficient during 15-day cultivation in this study (Table 1). Why the carbohydrate decreased while lipid increased in later phase of cultivation under nitrogen-replete condition will be observed in further study. On the other hand, potential competition between synthesis of lipid and carbohydrates is an important factor when deciding optimal biofuel production strategies. Microalgae lipid is used for biodiesel production while carbohydrate are excellent substrate for bioethanol production [26]. Tribonema sp. based carbohydrates are mainly in the form of cellulose (with the absence of lignin) are thus much easier to convert to monosaccharides when compared with lignocellulosic materials [27]. Therefore, Tribonema sp. biomass contains abundant carbohydrate could be considered for bioethanol fermentation, while biomass contains lipid for biodiesel production. Lipid composition and fatty acid profiles Triacylglycerols (TAG) are stored in specialized cytosolic oil bodies and function as energy reserve. Therefore, quantities and fatty acid compositions of triacylglycerols (TAG) in tested stains are important factors to evaluate
Fig. 4 Lipid compositions of tested microalgal strains cultured for 14 days
whether they can serve as feedstock for biodiesel production [28]. Lipid compositions of tested microalgae strains cultured by day 14 were analyzed using TLC-FID (Fig. 4). Sterol esters (SE), triacylglycerols (TAG), digalactosyl diacyglycerol (DAG), and phospholipid (PL) were presented in tested strains. TAGs accounted for 66.2, 78.9, 71.1, 75.1, 81.3 and 81.4 % of dry weights in T. vulgare 24.94, T. aequale 200.80, T. aequale 880-1, T. ulotrichoides 21.94, T. utriculosum 22.94 and T. minus 880-3, respectively. Obviously, TAG was the majority ingredient in total lipid in all tested strains under normal conditions, revealing the specie of Tribonema could be a potential raw material for biodiesel production. Phospholipids were the second ingredient in total lipids from tested strains, 15.1, 5.7, 22.2, 18.4, 15.4 and 12.5 % of phospholipids were detected in T. vulgare 24.94, T. aequale 200.80, T. aequale 880-1, T. ulotrichoides 21.94, T. utriculosum 22.94, T. minus 880-3, respectively. The lowest ratio of TAG/PL approached to 3.2 presented in T. aequale 880-1 yet the highest one accounted for 6.52 in T. minus 880-3. Such higher TAG/PL lipid predicts a relatively easier and better biodiesel conversion. Fatty acids in tested strains were primarily esterified and the major fatty acid composition of each was determined using GC–MS analysis (Table 2). The major fatty acids in the six strains were myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1), oleic acid (C18:1) comprising 6.9–12.1 %, 19.25–33.68 %, 38.49–51.21 % and 2.96–7.28 % of the total fatty acids, respectively, whereas myristoleic (C14:1), pentadecanoic (C15:0), hexadecadioneic acid (C16:2) and linoleic acid (C18:2) existed as minor fatty acids. Table 2 also revealed that saturated
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Bioprocess Biosyst Eng Table 2 Fatty acid composition of tested microalgal strains (% of total FAME) Properties
Strains T.v 24.94
T.a 200.80
T.a 880-1
T.u 21.94
T.u 22.94
T.m 880-3
C14:0
10.73
9.31
9.09
12.03
11.97
6.85
C14:1
0.62
nd
nd
0.54
0.98
nd
C15:0
0.46
nd
nd
0.86
1.07
0.56
C16:0
26.32
24.53
27.65
33.68
19.25
28.35
C16:1
42.5
51.1
45.93
38.49
51.21
50.65
C16:1
nd
nd
0.50
nd
nd
nd
C16:2
nd
0.72
0.58
0.68
nd
0.67
C16:2
nd
nd
nd
0.79
0.01
0.88
C18:0
1.72
0.66
1.06
1.2
0.99
1.02
C18:1
7.28
4.71
4.54
3.12
6.92
2.96
C18:2
0.63
0.55
0.65
nd
0.54
0.71
C20:3
1.82
1.56
1.75
1.43
1.74
1.20
C20:4
2.53
1.34
2.11
3.02
3.17
3.02
C20:5 SFA
5.32 39.23
5.45 35.56
6.12 37.81
4.15 46.78
2.15 33.29
3.14 36.78
MUFA
50.38
54.19
50.47
42.15
57.11
52.51
PUFA
10.3
9.62
11.21
10.7
7.61 ± 1.3
9.61
and monounsaturated fatty acids were the dominant components, comprising 88.28–90.4 % of the total fatty acids in the biodiesels from the tested algae. In addition, eicosapentaenoic acid (C20:5) was the main component of polyunsaturated fatty acids in all tested strains. Biodiesel reduces emissions of CO2, CO, hydrocarbons and particle emissions; however, such biodiesel also suffers from several performance-related problems including poor cold flow properties and insufficient oxidative stability [29]. The most common fatty acid methyl esters present in biodiesel are palmitic acid (16:0), steric acid (18:0), oleic acid (18:1), linoleic acid (18:2) and linolenic acid (18:3) [30]. However, palmitoleic acid (16:1) instead of linolenic acid (18:3) presented in the fatty acid profiles from six Tribonema strains. The monosaturated fatty acid was the dominant component, comprising 43.15–54.19 % of the total fatty acids in lipid from six strains, which was considered to be better than polyunsaturated fatty methyl esters for improving CN and oxidative stability without any concomitant adverse effect on the cold properties of the diesel [31].
Conclusion In short, all strains of genus Tribonema are unbranched filament composed of a single row of elongated, cylindrical cells, attaining 0.5–3 mm in length. The biomass productivities of all tested Tribonema sp. strains in bubbled
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column are ca. 0.35–0.42 g L-1 d-1, similar to those of unicellular microalgae in same condition. The genus Tribonema mainly accumulates carbohydrate in early phase and then induced lipid synthesis in later phase of cultivation under nitrogen repletion. The highest total lipid contents of tested strains ranged from 38 to 61 %, of which TAG was the majority ingredient. Besides the dominant components of palmitic acid (C16:0) and palmitoleic acid (C16:1), eicosapentaenoic acid (C20:5) was also found in all tested strains. Analyses of the present results suggest that genus Tribonema is promising feedstock for bioethanol and biodiesel production and such filamentous microalgae should be paid more attention. Acknowledgments This work was supported by the Solar Energy Initiative Plan (KGCX2-EW-309) of Chinese Academy of Sciences, Director Innovation Foundation of Qingdao Institute of Bioenergy and Bioprocess Technology, CAS (Y37204110E) and the Science and Technology Development Planning of Shandong Province (2013GGF01008).
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