Bioresource Technology 158 (2014) 19–24
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Special biochemical responses to nitrogen deprivation of filamentous oleaginous microalgae Tribonema sp. Fajin Guo a,b, Hui Wang a,⇑, Junfeng Wang a, Wenjun Zhou a, Lili Gao a, Lin Chen a, Qingzhe Dong a, Wei Zhang a, Tianzhong Liu a,⇑ a b
CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, PR China University of Chinese Academy of Sciences, Beijing 100049, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Effect of nitrogen on filamentous
microalgae Tribonema was tested for first time. Nitrogen starvation decrease the lipid and TAG contents of dry weight in Tribonema. Cytoplasm of Tribonema in any culture condition contains no starch granules. Nitrogen starvation trigger the accumulation mainly in insoluble sugar.
a r t i c l e
i n f o
Article history: Received 18 December 2013 Received in revised form 24 January 2014 Accepted 27 January 2014 Available online 10 February 2014 Keywords: Tribonema Nitrogen starvation Lipid Carbohydrate Fatty acid
a b s t r a c t Both filamentous microalgae Tribonema and unicellular microalgae Nanochloropsis are promising feedstock for biodiesel production. Nitrogen starvation increased lipid content in Nannochloropsis but decreased that in Tribonema. In this study, biochemical responses of Tribonema under different levels of nitrogen (0N, 0.05N, 0.1N and 1N-BG11) were investigated. 1N-BG11 was sufficient during 15-day-cultivation, while the other levels were nitrogen limited. Cell growth was interrupted with 0N-BG11, but no differences in biomass among 0.05N, 0.1N and 1N-BG11. Both protein and lipid contents (% of dry weight) declined gradually inversely to the increment in carbohydrate contents under the decreasement of nitrogen levels. Both assays and TEM results showed that the cytoplasm in Tribonema contained no starch. Compared to nitrogen-replete condition, the TAG content (% of dry weight) decreased obviously under nitrogen starvation. Different levels of nitrogen did not cause fundamental shifts in fatty acid profiles in Tribonema. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Alternative and ecologically friendly energy resource becomes increasingly important as the rapidly depleting of fossil fuels and the worsening environment (William and Laurens, 2010). Microalgae are of particular interests as a most promising source
⇑ Corresponding authors. Tel./fax: +86 53280662735. E-mail addresses: [email protected] (H. Wang), [email protected] (T. Liu). http://dx.doi.org/10.1016/j.biortech.2014.01.144 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
of biomass for biodiesel and jet fuel production due to their potential to synthesize and accumulate large quantities of lipids and higher growth rate compared to terrestrial plants (Ahmad et al., 2011; Chisti, 2007; Griffiths and Harrison, 2009; Hu et al., 2008). However, the production of biodiesel from microalgae is still too expensive to meet the market requirements (Acién et al., 2012). Harvesting is estimated to account for 20–30% of the total costs of microalgae biomass production (Mata et al., 2010). Moreover, the unicellular oleaginous algal cells are usually palatable for grazers to cause the crash of mass cultivation, and thus indirectly
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causing high costs (Wang et al., 2013a,b,c). Microalgae with bigger cell size might have potential in resisting the predation of grazers and decreasing the harvesting cost. Previous studies confirmed the hypothesis and successfully found a strain of filamentous oleaginous microalgae Tribonema sp. which could be used as biodiesel source (Wang et al., 2013a,b,c). Filamentous microalgae Tribonema sp. has unbranched filaments composed of a single row of elongated, cylindrical cells, it was relatively common in freshwater environments worldwide (Machova et al., 2007). Few attentions were paid on this genus until it was found to accumulate large quantity lipid droplets in cytoplasm (Wang et al., 2013a,b,c). While filamentous oleaginous microalgae specie Tribonema sp. has emerged as a potential microalgal energy source and the integrated process of biodiesel production from Tribonema sp. has been established, as a new filamentous oleaginous microalgae genus different from common unicellular microalgae, knowledge on regulating biochemical composition and increasing lipid especially triacylglycerol (TAG) on Tribonema needs to be improved for more efficient usage. Changes in environmental conditions such as temperature, light intensity and nutrient media characteristics are known to influence biochemical compositions of microalgae (Vince et al., 2012). Under optimal growth conditions, the lipid content of microalgae is approximately 10–30% dry weight, while under stressed conditions such as nutrient deprivation and high light intensity, the lipid content can double or triple, with up to 60–80% of dry weight recorded in some oleaginous species (Schenk et al., 2008; Vince et al., 2012). Among the above conditions, nitrogen starvation is the most widely used strategy for lipid induction of microalgae (Jiang et al., 2012). Nitrogen is a major constituent of proteins and nucleic acids. Accordingly, pigment and proteins losses are bound to trigger the accumulation of carbohydrate or lipid. Many researchers have proved that carbohydrate and lipid accumulated in different stage of the cultivation for most of the oleaginous microalgae species. For example, Turgay et al. (2012) reported that starch content increment was faster and higher than lipid increment in response to nitrogen starvation in Chlamydomonas reinhardtii. And there is also a competition or reciprocal transformation between the carbohydrate and lipid in microalgae (Li et al., 2010) under the nitrogen starvation. It should be noted that nitrogen deficiency is known to induce a wide variety of cellular response mechanism in living organism, yet the response varies from species to species. Tribonema sp. is the first filamentous specie of interest in biodiesel production due to its high lipid content, good insect resistance and relatively easy recovery. The aim of the present study was to testify if nitrogen deprivation could enhance the lipid accumulation and quantify some biochemical changes of Tribonema sp. grown in nitrogen-limited conditions.
2. Methods 2.1. Microalgae and culture conditions Besides filamentous microalgae Tribonema sp., Nannochloropsis sp., an unicellular oleaginous microalgae was also involved in this study to compare the effect of nitrogen starvation on the lipid accumulations in two microalgae. Fresh microalgae Tribonema sp. (purchased from SAG culture collection, University of Gottingen, Germany), were maintained in BG11 medium (Boussiba and Vonshak, 1991). The marine specie Nannochloropsis OZ-1, courtesy of Dr. Feng Chen of the University of Maryland Center for Environmental Science, was maintained in artificial sea water enriched with full strength of BG11 nutrients.
Columns (80 cm height and 5 cm diameter) with 700 mL of media were used to cultivate the two microalgae species. Compressed air containing 1.5% CO2 was continuously pressed to mix the cultivation medium and supply the carbon source. Temperature was maintained at 24 ± 1 °C. The light source was provided by the fluorescent tubes, which were placed horizontally and parallelly to the front side of columns. During the cultivation, the light was on continuously and light intensity was 120 ± 5 lmol photons m 2 s 1. 2.2. Experimental design Two investigations were designed and involved in this research. 2.2.1. Comparison of the effects on lipid accumulation in Tribonema and Nannochloropsis under nitrogen starvation Cells from cultures of both Tribonema and Nannochloropsis were grown n nitrogen sufficient medium for 5 days and then pelleted by suction filtration, washed twice with deionized water and ressuspended into nitrogen-deplete BG11 medium for another 5 days. Culture lipid contents were measured via gravimetric method (see below) every day. 2.2.2. Growth and Biochemical response to different levels of nitrogen in Tribonema Cultures (700 mL) of Tribonema culture were grown under different levels of sodium nitrate supplementations (0, 0.075, 0.15, 0.3, 0.75, 1.5 g/L) for 15 days, where samples were taken every 3 days for nutrients, cell growths, proteins, carbohydrates and lipids. In addition, triacylglycerols (TAGs) content and fatty acid profiles were analyzed at the end of the cultivation. 2.3. Analytical methods For nutrient analyses, 10 mL samples of culture were filtered (0.2 lm), the filtrate was stored in 20 °C freezer to await analysis. After thawing, samples were analyzed for concentrations using a nutrient autoanalyser (Vario EL cube, Elementary Inc., Germany) employing the method from Brewer and Riley (1965). A certain volume of microalgae (V) culture was filtered to preweighted 0.45 lm GF/C filter membrane (Whatman, DW0). The membrane was oven dried at 105 °C overnight and then weighted (DW1). The biomass density of culture was calculated as (DW1–DW0)/V. Protein extraction was performed according to Turgay et al. (2012). Freeze-dried cells pellets were re-suspended in lysis buffer (50 mM Tris–HCl pH 8.0, 2% SDS, 10 mM EDTA, protease inhibitor mix), subjugated to sonication for 1 min at 60% powder, frozen in liquid nitrogen for 1 min, thawed and centrifuged at 13,000g for 20 min at 4 °C. The supernatant was used for protein determination with Bradford method (Bradford, 1976). To assess the carbohydrate composition and content in microalgae, soluble sugar, starch and cellulose were separated firstly as described by Wang et al. (2013a,b,c), following which, the contents of carbohydrates were determined using the modified quantitative scarification (QS) method reported by the National Renewable Energy Laboratory (NREL),USA (Moxley and Zhang, 2007). Total lipid content was determined by gravimetric analysis according to Chen et al. (2012) with some modifications. Approximately 50 mg of dried algae pellet was grinded with quartz firstly and then mixed with 7.5 ml methanol/chloroform (2:1, v/v) at 37 °C overnight. The mixture was then centrifuged at 8000g for 10 min, and the supernatant was collected and residual biomass was extracted one more time. 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). The
F. Guo et al. / Bioresource Technology 158 (2014) 19–24
organic phase was carefully transferred to a vial and dried to constant weight with nitrogen flow. The total lipid content was calculated as a percentage of the dry weight of the algae. Triacylglycerol (TAG) content in lipid was analyzed using a thinlayer chromatography coupled with flame ionization detection system (TLC–FID, MK-6, latron laboratories, Inc., Japan) (Fedosov et al., 2011). The individual lipid component was identified by co-chromatography with pure standards (SE&HC; FAME; TAG; DAG; MAG&PL, purchased from Sigma, Louis, MO. USA). Fatty acid profiles were determined post-conversion to fatty acid methyl esters (FAMEs) and analyzed on a Varian 450GC (Varian Inc., USA). Lipid was converted to FAMEs with H2SO4/methanol (1:50, w/v) and nonadecanoic acid (C19:0) was added as an internal standard, which was followed by incubation at 85 °C for 2.5 h. Nitrogen was used as carrier gas during FAME analysis on 450GC, the injector temperature was set as 280 °C with an injection volume of 2 lL under split mode (10:1). The individual FAMEs were identified by chromatographic comparison with authentic standards (Sigma) and the quantities of individual FAMEs were calculated as reports by Chen et al. (2012). Microalgae cells were observed using transmission electron microscopy (TEM). Algae cells were once fixed by 1% glutaraldehyde for 1 h and twice by 1% osimic acid for 1 h and after each fixation cells were washed with phosphate buffered saline (PBS). The samples were dehydrated through an acetone series (30– 90%), followed by pure acetone for three times. The samples were permeate and embedded with acetone-Spurr’s resin mixture for 5 h, acetone-Spurr’s resin mixture overnight and Spurr’s resin for 5 h. The embedded samples were polymerized at 65 °C for 24 h. The ultrathin sections were examined with a Hitachi H-7650 transmission electron microscope (Hitachi High-Technologies Co., Tokyo, Japan).
3. Results and discussion 3.1. Effect of nitrogen starvation on lipid accumulation in Tribonema and Nannochloropsis The lipid contents of Tribonema and Nannochloropsis in nitrogen sufficient (denoted as 1N) and nitrogen free (denoted as 0N) media were shown in Fig. 1. In previous 5-day-cultivation of both microalgae species in nitrogen sufficient medium, the lipid contents in both species slightly changed because the carbon source absorbed by microalgae was mainly used for biomass increment. Afterwards, half culture of each species were pelleted by suction filtration, washed twice with deionized water and res-suspended into nitrogen-free BG11 medium for another 5 days, while the other half culture of each species still remained in nitrogen sufficient medium. For Nannochloropsis cultures, the total amount of cellular lipid (ca. 43.04%) was significantly higher in 5-day nitrogen-free cultivation than that (ca. 26.16%) in nitrogen-replete medium. This was consistent with the reports in Chaetoceros muelleri (Bacillariphyceae) and Dunaliella salina (Chlorophyceae), the lipid contents of which reached 46.32% and 47.34% in nitrogen deprivation medium compared to 22.74% and 26.37% in nitrogen sufficient medium (Gao et al., 2013). However, for Tribonema, the lipid content increased from ca. 29.64% to ca. 49.55% (of dry weight) in nitrogen-replete medium, while lipid content only reached 39.47% under nitrogen-free condition. Nitrogen is the most important element contributing to microalgae cells, its deprivation significantly changed the physiological and biochemical parameters (Jiang et al., 2012). As protein is a nitrogen-rich compound, nitrogen starvation can reduce protein content, extra energy flow would shift to carbohydrate and lipid, and there was a competition between these two components.
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Fig. 1. Cellular lipid contents (% of dry weight) in Tribonema (A) and Nannochloropsis (B) in both nitrogen-replete and nitrogen-free medium, respectively.
Interestingly, nitrogen starvation resulted in the increment of lipid accumulation in Nannochloropsis sp., but decrement of lipid accumulation in Tribonema sp., suggesting that the lipid accumulation metabolism in filamentous microalgae Tribonema was special and different from common oleaginous microalgae. Therefore, growth, biochemical change and biodiesel feedstock production under different levels of nitrogen were further investigated as follows to clarify the carbon flow problem in filamentous microalgae Tribonema sp. 3.2. Nitrate utilization and growth response to different levels of nitrogen in Tribonema Cells of Tribonema were grown in BG11 medium with different levels of nitrogen supplementation during 15 days. The standard BG11 medium with NaNO3 of 1.5 g/L was regarded as control (1N), the other NaNO3 levels of 0 g/L, 0.075 g/L, 0.15 g/L, 0.3 g/L, 0.75 g/L in BG11 medium were referred to as 0N, 0.05N, 0.1N, 0.2N, 0.5N, respectively, for convenience. The variations of N NO3 concentration in the media during the cultivations were shown in Fig. 2A. The results clearly demonstrated that the concentrations of N NO3 decreased sharply in 6-day-cultivation in 1NBG11 and 0.5N-BG11 medium and then maintained ca. 156 mg/L and ca. 22 mg/L, respectively, till the end of the cultivation, suggesting both 1N-BG11 and 0.5N-BG11 were nitrogen-replete medium. However, Fig. 2A also revealed that nitrogen in other initial levels (0.2N, 0.1N and 0.5N) was completely consumed at day 3, which meant initial 0.2N-BG11, 0.1N-BG11 and 0.5N-BG11 were nitrogen-limited medium. The growth of Tribonema in BG11 medium with different nitrogen levels were shown in Fig. 2B. Microalgae had exponential growth phase followed by the late logarithmic phase at day 12 in BG11 medium with highest nitrogen levels (except 0N). The nitrogen deprivation significantly reduced the biomass of Tribonema, which was in agreement with other reports (Chen et al., 2011). The highest biomass of microalgae in nitrogen-free medium reached 1.16 g/L by day 3. Afterwards, there was a slight decrement in biomass. Biomass of microalgae cultured in 0.05N-BG11
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Fig. 2. Nitrogen concentrations in the culture (A) and biomass of Tribonema (B) under different levels of nitrogen supply.
medium reached 3.82 g/L in 15-day-cultivation. However, there was no obvious difference of biomass density among the cultures of other nitrogen levels (0.1N, 0.2N, 0.5N and 1N). The final biomass density of all these treatments accounted for ca. 4.71– 4.93 g/L. Though nitrogen deprivation occurred after the third or sixth day of the cultures with 0.1N and 0.2N-BG11 (Fig. 2A), the biomass densities of microalgae cultured in 0.1N and 0.2N-BG11 medium were the same as that in 1N-BG11 medium. A reasonable explanation might be that Tribonema absorbed the nitrogen fast in the initial stage of cultivation, or used some other nitrogen sources such as the cellular organic nitrogen, to maintain its growth. According to the nitrogen consumption and growth condition, nitrogen-replete medium (1N-BG11), nitrogen-limited medium (0.1N-BG11, 0.05N-BG11) and nitrogen-free medium (0N-BG11) were adopted to study the biochemical response and fatty acid profiles of Tribonema. 3.3. Biochemical response to different levels of nitrogen in Tribonema Total protein contents in microalgae samples decreased drastically in response to all nitrogen levels (1N, 0.1N, 0.0.05N, 0N) during the cultivation period (Fig. 3A). A 77.02% decrease in protein was observed after 3 days of nitrogen-free and there was a slight decrease from day 3 to day 15. In nitrogen replete medium (1NBG11), protein increased at the start of the experiment (day 3) in response to the Tribonema being placed in fresh growth medium. Afterwards, the protein decreased continuously to 12.65% (% of dry weight) at day 15, but it was still higher than that (ca. 6.8% of dry weight) in nitrogen-free medium. On the other hand, total lipid contents in microalgae with all treatments had different
degrees of decrement by day 3, followed by significant increment from day 3 to day 15 (Fig. 3B). The final lipid contents with 0N, 0.05N, 0.1N and 1N were 45.59%, 48.16%, 52.28% and 57.02%, respectively. Maximum lipid content was observed in microalgae in 1N-BG11 medium, which was in accordance with the result of the above first investigation. The result confirmed again that nitrogen starvation decreased the lipid content in filamentous microalgae Tribonema. Corresponding methods were adopted to detect the contents and compositions of carbohydrate in Tribonema with all treatments. Interestingly, no starch was detected in microalgae cells anytime. To verify this, images of transmission electron microscopy of microalgae Tribonema were observed. Tribonema had unbranched filaments composed of a single row of elongated, cylindrical cells. The cell wall is made up of double cylinders that overlap to enclose the cell content, and these wall pieces appear H-shaped. The cylindrical cells were 3–5 lm wide and 15–25 lm long. Chloroplasts, the most obvious organelle, piled up mainly in both sides in cells. And the cytoplasm contains obvious lipid droplet, but no starch granules can be observed. The soluble sugar (monosaccharide and disaccharide) and insoluble sugar (mainly cellulose) contents were calculated based on biomass (Fig. 3C and D). Soluble sugar contents (% of dry weight) in microalgae with all levels of nitrogen increased by day 3 and then decreased to almost the same level (3.76% of dry weight) till the end of cultivation. Among all the conditions, insoluble sugar contents (% of dry weight) increased in different degree, followed by decrement from day 6 to day 15 (Fig. 3D). The final insoluble sugar content was 30.57% under initial nitrogen free stress, while that was 18.29% in nitrogen-replete medium (1N-BG11). Therefore, it can be concluded that for Tribonema sp., there was not a starch synthesis mechanism in the cytoplasm, and nitrogen starvation triggered the carbohydrate accumulation, mainly cellulose, while decreased the lipid content (% of dry weight). In addition, potential competition plays an important role in the carbon flux between synthesis of lipid and carbohydrates (Turgay et al., 2012). According to the data in Fig. 3, under all treatments, carbohydrate was the dominated components in mid-term of the cultivation, while lipid was the main component in late stage of cultivation. Therefore, there are two strategies for bioenergy production for Tribonema sp., short-term cultivation for ethanol fermentation and long-term cultivation for biodiesel production, due to the dominant components are carbohydrate and lipid, respectively.
3.4. Triacylglycerols (TAGs) accumulation to different levels of nitrogen in Tribonema Many investigations focused on the impact of nitrogen starvation on total lipid accumulation whereas only a few reports are available about the impact of nitrogen limitation on TAG accumulation. Triacylglycerols (TAGs) are stored in specialized cytosolic oil bodies and function as energy reserve, and more desirable than phospholipids and glycolipids for biodiesel production as they have a higher FA (fatty acid) content and no phosphate (Mata et al., 2010; Miao and Wu, 2006). TAGs contents of Tribonema in different nitrogen levels medium at the end of cultivation were analyzed using TLC–FID (Table 1). In contrast to the impact on the biomass and total lipid content, slight differences were observed in the TAG contents (% of lipid) in microalgae cultured with different levels of nitrogen. TAG was the majority composition in total lipid with all treatments, accounted for ca. 82.77–87.5% (of total lipid). However, significant distinctions existed among the TAG contents in dry weight, as the lipid contents of dry weight linearly decreased from high levels to low levels of nitrogen. The final TAG
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Fig. 3. Dynamics of cellular components, protein (A), lipid (B), soluble sugar (C) and insoluble sugar (D) in Tribonema, in response to 0N, 0.05N, 0.1N and 1N-BG11 medium.
Table 1 TAG contents in microalgae with different levels of nitrogen supply. Nitrogen concentration
TAG (% of lipid) TAG (% of dry weight)
0N-BG11
0.05NBG11
0.1NBG11
1N-BG11
82.77 ± 1.2 37.74 ± 0.3
83.96 ± 0.9 40.45 ± 2.4
86.31 ± 1.7 45.13 ± 1.1
87.5 ± 4.3 49.89 ± 2.3
contents of dry weight were 37.74%, 40.45%, 45.13% and 49.89%, respectively, in response to 0N, 0.05N, 0.1N and 1N-BG11. To our knowledge, in general, a common practice to stimulate TAG accumulation in microalgae is to cultivate microalgae longer to produce a natural nitrogen stress environments by the gradually used up of nitrogen as this way is cost effective and easy to manipulate (Brennan and Qwende, 2010). The total fatty acid content has a minor decrease upon nitrogen starvation,which agreed with those by Dunaliella tertiolecta (Guido et al., 2013), though the reasons are unknown. 3.5. Fatty acid profiles of Tribonema under different nitrogen levels In addition to TAGs content, fatty acid profile of microalgae is also an important characteristic as it ultimately affects the quality of the biodiesel product (Gouveia and Oliveira, 2009; Knothe, 2005). Fatty acid profiles of Tribonema were determined at the end of cultivation (day 15) under all nitrogen levels (Fig. 4). Among all conditions, palmitoleic acid (C16:1) was always the dominant fatty acid, constituting up to ca. 49.09–59.37% of total fatty acids in Tribonema, while oleic acid was in majority in green algae (Griffiths et al., 2011). The second main composition was palmitic acid (C16:0). However, the oleic acid (C18:1) in Tribonema only accounted for 3.94%, 3.81%, 3.7%, and 2.49%, respectively, in response to 0N, 0.05N, 0.1N and 1N-BG11 medium. Furthermore, eicosapentaenoic (C20:5) existed among all treatments, the contents were from 2.58% to 2.93% (% of total fatty acid). In total, fatty acid compositions upon all levels of nitrogen were very similar, though 8.4% of myristic acid (C14:0) and 49.1% of
Fig. 4. Fatty acid profiles of Tribonema with different levels of nitrogen supply.
palmitoleic (C16:1) were observed in nitrogen-free medium (0N-BG11) while those of 5.16% and 56.66%, respectively in nitrogen-replete medium (1N-BG11). The result meant that different nitrogen levels did not cause fundamental shifts in fatty acid compositions of lipid from filamentous microalgae Tribonema.
4. Conclusion Filamentous oleaginous microalgae Tribonema is promising feedstock for biofuel production. However, it had different biochemical response compared with general unicellular oleaginous microalgae. There is no starch synthesis pathway in the Tribonema cells. Carbohydrate (cellulose) was the dominated component in mid-term of the cultivation, while lipid was the main component in late stage of cultivation. In addition, total protein and TAG(s) contents (% of dry weight) decreased in response to the increment of carbohydrate (mainly cellulose) in Tribonema cells under nitrogen starvation conditions. Therefore, further research on the
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correlative relationship between synthesis of carbohydrate and lipid in Tribonema is recommended.
Acknowledgements This work was supported by Qibebt (CAS)-Boeing Joint Research Laboratory for Sustainable Aviation Biofuel, the Director Innovation Foundation of Qibebt (CAS) and Science and the technology development plan of Shandong Province (2013GGF01008).
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Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Special biochemical responses to nitrogen deprivation of filamentous oleaginous microalgae Tribonema sp. Fajin Guo a,b, Hui Wang a,⇑, Junfeng Wang a, Wenjun Zhou a, Lili Gao a, Lin Chen a, Qingzhe Dong a, Wei Zhang a, Tianzhong Liu a,⇑ a b
CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, PR China University of Chinese Academy of Sciences, Beijing 100049, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Effect of nitrogen on filamentous
microalgae Tribonema was tested for first time. Nitrogen starvation decrease the lipid and TAG contents of dry weight in Tribonema. Cytoplasm of Tribonema in any culture condition contains no starch granules. Nitrogen starvation trigger the accumulation mainly in insoluble sugar.
a r t i c l e
i n f o
Article history: Received 18 December 2013 Received in revised form 24 January 2014 Accepted 27 January 2014 Available online 10 February 2014 Keywords: Tribonema Nitrogen starvation Lipid Carbohydrate Fatty acid
a b s t r a c t Both filamentous microalgae Tribonema and unicellular microalgae Nanochloropsis are promising feedstock for biodiesel production. Nitrogen starvation increased lipid content in Nannochloropsis but decreased that in Tribonema. In this study, biochemical responses of Tribonema under different levels of nitrogen (0N, 0.05N, 0.1N and 1N-BG11) were investigated. 1N-BG11 was sufficient during 15-day-cultivation, while the other levels were nitrogen limited. Cell growth was interrupted with 0N-BG11, but no differences in biomass among 0.05N, 0.1N and 1N-BG11. Both protein and lipid contents (% of dry weight) declined gradually inversely to the increment in carbohydrate contents under the decreasement of nitrogen levels. Both assays and TEM results showed that the cytoplasm in Tribonema contained no starch. Compared to nitrogen-replete condition, the TAG content (% of dry weight) decreased obviously under nitrogen starvation. Different levels of nitrogen did not cause fundamental shifts in fatty acid profiles in Tribonema. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Alternative and ecologically friendly energy resource becomes increasingly important as the rapidly depleting of fossil fuels and the worsening environment (William and Laurens, 2010). Microalgae are of particular interests as a most promising source
⇑ Corresponding authors. Tel./fax: +86 53280662735. E-mail addresses: [email protected] (H. Wang), [email protected] (T. Liu). http://dx.doi.org/10.1016/j.biortech.2014.01.144 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
of biomass for biodiesel and jet fuel production due to their potential to synthesize and accumulate large quantities of lipids and higher growth rate compared to terrestrial plants (Ahmad et al., 2011; Chisti, 2007; Griffiths and Harrison, 2009; Hu et al., 2008). However, the production of biodiesel from microalgae is still too expensive to meet the market requirements (Acién et al., 2012). Harvesting is estimated to account for 20–30% of the total costs of microalgae biomass production (Mata et al., 2010). Moreover, the unicellular oleaginous algal cells are usually palatable for grazers to cause the crash of mass cultivation, and thus indirectly
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causing high costs (Wang et al., 2013a,b,c). Microalgae with bigger cell size might have potential in resisting the predation of grazers and decreasing the harvesting cost. Previous studies confirmed the hypothesis and successfully found a strain of filamentous oleaginous microalgae Tribonema sp. which could be used as biodiesel source (Wang et al., 2013a,b,c). Filamentous microalgae Tribonema sp. has unbranched filaments composed of a single row of elongated, cylindrical cells, it was relatively common in freshwater environments worldwide (Machova et al., 2007). Few attentions were paid on this genus until it was found to accumulate large quantity lipid droplets in cytoplasm (Wang et al., 2013a,b,c). While filamentous oleaginous microalgae specie Tribonema sp. has emerged as a potential microalgal energy source and the integrated process of biodiesel production from Tribonema sp. has been established, as a new filamentous oleaginous microalgae genus different from common unicellular microalgae, knowledge on regulating biochemical composition and increasing lipid especially triacylglycerol (TAG) on Tribonema needs to be improved for more efficient usage. Changes in environmental conditions such as temperature, light intensity and nutrient media characteristics are known to influence biochemical compositions of microalgae (Vince et al., 2012). Under optimal growth conditions, the lipid content of microalgae is approximately 10–30% dry weight, while under stressed conditions such as nutrient deprivation and high light intensity, the lipid content can double or triple, with up to 60–80% of dry weight recorded in some oleaginous species (Schenk et al., 2008; Vince et al., 2012). Among the above conditions, nitrogen starvation is the most widely used strategy for lipid induction of microalgae (Jiang et al., 2012). Nitrogen is a major constituent of proteins and nucleic acids. Accordingly, pigment and proteins losses are bound to trigger the accumulation of carbohydrate or lipid. Many researchers have proved that carbohydrate and lipid accumulated in different stage of the cultivation for most of the oleaginous microalgae species. For example, Turgay et al. (2012) reported that starch content increment was faster and higher than lipid increment in response to nitrogen starvation in Chlamydomonas reinhardtii. And there is also a competition or reciprocal transformation between the carbohydrate and lipid in microalgae (Li et al., 2010) under the nitrogen starvation. It should be noted that nitrogen deficiency is known to induce a wide variety of cellular response mechanism in living organism, yet the response varies from species to species. Tribonema sp. is the first filamentous specie of interest in biodiesel production due to its high lipid content, good insect resistance and relatively easy recovery. The aim of the present study was to testify if nitrogen deprivation could enhance the lipid accumulation and quantify some biochemical changes of Tribonema sp. grown in nitrogen-limited conditions.
2. Methods 2.1. Microalgae and culture conditions Besides filamentous microalgae Tribonema sp., Nannochloropsis sp., an unicellular oleaginous microalgae was also involved in this study to compare the effect of nitrogen starvation on the lipid accumulations in two microalgae. Fresh microalgae Tribonema sp. (purchased from SAG culture collection, University of Gottingen, Germany), were maintained in BG11 medium (Boussiba and Vonshak, 1991). The marine specie Nannochloropsis OZ-1, courtesy of Dr. Feng Chen of the University of Maryland Center for Environmental Science, was maintained in artificial sea water enriched with full strength of BG11 nutrients.
Columns (80 cm height and 5 cm diameter) with 700 mL of media were used to cultivate the two microalgae species. Compressed air containing 1.5% CO2 was continuously pressed to mix the cultivation medium and supply the carbon source. Temperature was maintained at 24 ± 1 °C. The light source was provided by the fluorescent tubes, which were placed horizontally and parallelly to the front side of columns. During the cultivation, the light was on continuously and light intensity was 120 ± 5 lmol photons m 2 s 1. 2.2. Experimental design Two investigations were designed and involved in this research. 2.2.1. Comparison of the effects on lipid accumulation in Tribonema and Nannochloropsis under nitrogen starvation Cells from cultures of both Tribonema and Nannochloropsis were grown n nitrogen sufficient medium for 5 days and then pelleted by suction filtration, washed twice with deionized water and ressuspended into nitrogen-deplete BG11 medium for another 5 days. Culture lipid contents were measured via gravimetric method (see below) every day. 2.2.2. Growth and Biochemical response to different levels of nitrogen in Tribonema Cultures (700 mL) of Tribonema culture were grown under different levels of sodium nitrate supplementations (0, 0.075, 0.15, 0.3, 0.75, 1.5 g/L) for 15 days, where samples were taken every 3 days for nutrients, cell growths, proteins, carbohydrates and lipids. In addition, triacylglycerols (TAGs) content and fatty acid profiles were analyzed at the end of the cultivation. 2.3. Analytical methods For nutrient analyses, 10 mL samples of culture were filtered (0.2 lm), the filtrate was stored in 20 °C freezer to await analysis. After thawing, samples were analyzed for concentrations using a nutrient autoanalyser (Vario EL cube, Elementary Inc., Germany) employing the method from Brewer and Riley (1965). A certain volume of microalgae (V) culture was filtered to preweighted 0.45 lm GF/C filter membrane (Whatman, DW0). The membrane was oven dried at 105 °C overnight and then weighted (DW1). The biomass density of culture was calculated as (DW1–DW0)/V. Protein extraction was performed according to Turgay et al. (2012). Freeze-dried cells pellets were re-suspended in lysis buffer (50 mM Tris–HCl pH 8.0, 2% SDS, 10 mM EDTA, protease inhibitor mix), subjugated to sonication for 1 min at 60% powder, frozen in liquid nitrogen for 1 min, thawed and centrifuged at 13,000g for 20 min at 4 °C. The supernatant was used for protein determination with Bradford method (Bradford, 1976). To assess the carbohydrate composition and content in microalgae, soluble sugar, starch and cellulose were separated firstly as described by Wang et al. (2013a,b,c), following which, the contents of carbohydrates were determined using the modified quantitative scarification (QS) method reported by the National Renewable Energy Laboratory (NREL),USA (Moxley and Zhang, 2007). Total lipid content was determined by gravimetric analysis according to Chen et al. (2012) with some modifications. Approximately 50 mg of dried algae pellet was grinded with quartz firstly and then mixed with 7.5 ml methanol/chloroform (2:1, v/v) at 37 °C overnight. The mixture was then centrifuged at 8000g for 10 min, and the supernatant was collected and residual biomass was extracted one more time. 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). The
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organic phase was carefully transferred to a vial and dried to constant weight with nitrogen flow. The total lipid content was calculated as a percentage of the dry weight of the algae. Triacylglycerol (TAG) content in lipid was analyzed using a thinlayer chromatography coupled with flame ionization detection system (TLC–FID, MK-6, latron laboratories, Inc., Japan) (Fedosov et al., 2011). The individual lipid component was identified by co-chromatography with pure standards (SE&HC; FAME; TAG; DAG; MAG&PL, purchased from Sigma, Louis, MO. USA). Fatty acid profiles were determined post-conversion to fatty acid methyl esters (FAMEs) and analyzed on a Varian 450GC (Varian Inc., USA). Lipid was converted to FAMEs with H2SO4/methanol (1:50, w/v) and nonadecanoic acid (C19:0) was added as an internal standard, which was followed by incubation at 85 °C for 2.5 h. Nitrogen was used as carrier gas during FAME analysis on 450GC, the injector temperature was set as 280 °C with an injection volume of 2 lL under split mode (10:1). The individual FAMEs were identified by chromatographic comparison with authentic standards (Sigma) and the quantities of individual FAMEs were calculated as reports by Chen et al. (2012). Microalgae cells were observed using transmission electron microscopy (TEM). Algae cells were once fixed by 1% glutaraldehyde for 1 h and twice by 1% osimic acid for 1 h and after each fixation cells were washed with phosphate buffered saline (PBS). The samples were dehydrated through an acetone series (30– 90%), followed by pure acetone for three times. The samples were permeate and embedded with acetone-Spurr’s resin mixture for 5 h, acetone-Spurr’s resin mixture overnight and Spurr’s resin for 5 h. The embedded samples were polymerized at 65 °C for 24 h. The ultrathin sections were examined with a Hitachi H-7650 transmission electron microscope (Hitachi High-Technologies Co., Tokyo, Japan).
3. Results and discussion 3.1. Effect of nitrogen starvation on lipid accumulation in Tribonema and Nannochloropsis The lipid contents of Tribonema and Nannochloropsis in nitrogen sufficient (denoted as 1N) and nitrogen free (denoted as 0N) media were shown in Fig. 1. In previous 5-day-cultivation of both microalgae species in nitrogen sufficient medium, the lipid contents in both species slightly changed because the carbon source absorbed by microalgae was mainly used for biomass increment. Afterwards, half culture of each species were pelleted by suction filtration, washed twice with deionized water and res-suspended into nitrogen-free BG11 medium for another 5 days, while the other half culture of each species still remained in nitrogen sufficient medium. For Nannochloropsis cultures, the total amount of cellular lipid (ca. 43.04%) was significantly higher in 5-day nitrogen-free cultivation than that (ca. 26.16%) in nitrogen-replete medium. This was consistent with the reports in Chaetoceros muelleri (Bacillariphyceae) and Dunaliella salina (Chlorophyceae), the lipid contents of which reached 46.32% and 47.34% in nitrogen deprivation medium compared to 22.74% and 26.37% in nitrogen sufficient medium (Gao et al., 2013). However, for Tribonema, the lipid content increased from ca. 29.64% to ca. 49.55% (of dry weight) in nitrogen-replete medium, while lipid content only reached 39.47% under nitrogen-free condition. Nitrogen is the most important element contributing to microalgae cells, its deprivation significantly changed the physiological and biochemical parameters (Jiang et al., 2012). As protein is a nitrogen-rich compound, nitrogen starvation can reduce protein content, extra energy flow would shift to carbohydrate and lipid, and there was a competition between these two components.
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Fig. 1. Cellular lipid contents (% of dry weight) in Tribonema (A) and Nannochloropsis (B) in both nitrogen-replete and nitrogen-free medium, respectively.
Interestingly, nitrogen starvation resulted in the increment of lipid accumulation in Nannochloropsis sp., but decrement of lipid accumulation in Tribonema sp., suggesting that the lipid accumulation metabolism in filamentous microalgae Tribonema was special and different from common oleaginous microalgae. Therefore, growth, biochemical change and biodiesel feedstock production under different levels of nitrogen were further investigated as follows to clarify the carbon flow problem in filamentous microalgae Tribonema sp. 3.2. Nitrate utilization and growth response to different levels of nitrogen in Tribonema Cells of Tribonema were grown in BG11 medium with different levels of nitrogen supplementation during 15 days. The standard BG11 medium with NaNO3 of 1.5 g/L was regarded as control (1N), the other NaNO3 levels of 0 g/L, 0.075 g/L, 0.15 g/L, 0.3 g/L, 0.75 g/L in BG11 medium were referred to as 0N, 0.05N, 0.1N, 0.2N, 0.5N, respectively, for convenience. The variations of N NO3 concentration in the media during the cultivations were shown in Fig. 2A. The results clearly demonstrated that the concentrations of N NO3 decreased sharply in 6-day-cultivation in 1NBG11 and 0.5N-BG11 medium and then maintained ca. 156 mg/L and ca. 22 mg/L, respectively, till the end of the cultivation, suggesting both 1N-BG11 and 0.5N-BG11 were nitrogen-replete medium. However, Fig. 2A also revealed that nitrogen in other initial levels (0.2N, 0.1N and 0.5N) was completely consumed at day 3, which meant initial 0.2N-BG11, 0.1N-BG11 and 0.5N-BG11 were nitrogen-limited medium. The growth of Tribonema in BG11 medium with different nitrogen levels were shown in Fig. 2B. Microalgae had exponential growth phase followed by the late logarithmic phase at day 12 in BG11 medium with highest nitrogen levels (except 0N). The nitrogen deprivation significantly reduced the biomass of Tribonema, which was in agreement with other reports (Chen et al., 2011). The highest biomass of microalgae in nitrogen-free medium reached 1.16 g/L by day 3. Afterwards, there was a slight decrement in biomass. Biomass of microalgae cultured in 0.05N-BG11
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Fig. 2. Nitrogen concentrations in the culture (A) and biomass of Tribonema (B) under different levels of nitrogen supply.
medium reached 3.82 g/L in 15-day-cultivation. However, there was no obvious difference of biomass density among the cultures of other nitrogen levels (0.1N, 0.2N, 0.5N and 1N). The final biomass density of all these treatments accounted for ca. 4.71– 4.93 g/L. Though nitrogen deprivation occurred after the third or sixth day of the cultures with 0.1N and 0.2N-BG11 (Fig. 2A), the biomass densities of microalgae cultured in 0.1N and 0.2N-BG11 medium were the same as that in 1N-BG11 medium. A reasonable explanation might be that Tribonema absorbed the nitrogen fast in the initial stage of cultivation, or used some other nitrogen sources such as the cellular organic nitrogen, to maintain its growth. According to the nitrogen consumption and growth condition, nitrogen-replete medium (1N-BG11), nitrogen-limited medium (0.1N-BG11, 0.05N-BG11) and nitrogen-free medium (0N-BG11) were adopted to study the biochemical response and fatty acid profiles of Tribonema. 3.3. Biochemical response to different levels of nitrogen in Tribonema Total protein contents in microalgae samples decreased drastically in response to all nitrogen levels (1N, 0.1N, 0.0.05N, 0N) during the cultivation period (Fig. 3A). A 77.02% decrease in protein was observed after 3 days of nitrogen-free and there was a slight decrease from day 3 to day 15. In nitrogen replete medium (1NBG11), protein increased at the start of the experiment (day 3) in response to the Tribonema being placed in fresh growth medium. Afterwards, the protein decreased continuously to 12.65% (% of dry weight) at day 15, but it was still higher than that (ca. 6.8% of dry weight) in nitrogen-free medium. On the other hand, total lipid contents in microalgae with all treatments had different
degrees of decrement by day 3, followed by significant increment from day 3 to day 15 (Fig. 3B). The final lipid contents with 0N, 0.05N, 0.1N and 1N were 45.59%, 48.16%, 52.28% and 57.02%, respectively. Maximum lipid content was observed in microalgae in 1N-BG11 medium, which was in accordance with the result of the above first investigation. The result confirmed again that nitrogen starvation decreased the lipid content in filamentous microalgae Tribonema. Corresponding methods were adopted to detect the contents and compositions of carbohydrate in Tribonema with all treatments. Interestingly, no starch was detected in microalgae cells anytime. To verify this, images of transmission electron microscopy of microalgae Tribonema were observed. Tribonema had unbranched filaments composed of a single row of elongated, cylindrical cells. The cell wall is made up of double cylinders that overlap to enclose the cell content, and these wall pieces appear H-shaped. The cylindrical cells were 3–5 lm wide and 15–25 lm long. Chloroplasts, the most obvious organelle, piled up mainly in both sides in cells. And the cytoplasm contains obvious lipid droplet, but no starch granules can be observed. The soluble sugar (monosaccharide and disaccharide) and insoluble sugar (mainly cellulose) contents were calculated based on biomass (Fig. 3C and D). Soluble sugar contents (% of dry weight) in microalgae with all levels of nitrogen increased by day 3 and then decreased to almost the same level (3.76% of dry weight) till the end of cultivation. Among all the conditions, insoluble sugar contents (% of dry weight) increased in different degree, followed by decrement from day 6 to day 15 (Fig. 3D). The final insoluble sugar content was 30.57% under initial nitrogen free stress, while that was 18.29% in nitrogen-replete medium (1N-BG11). Therefore, it can be concluded that for Tribonema sp., there was not a starch synthesis mechanism in the cytoplasm, and nitrogen starvation triggered the carbohydrate accumulation, mainly cellulose, while decreased the lipid content (% of dry weight). In addition, potential competition plays an important role in the carbon flux between synthesis of lipid and carbohydrates (Turgay et al., 2012). According to the data in Fig. 3, under all treatments, carbohydrate was the dominated components in mid-term of the cultivation, while lipid was the main component in late stage of cultivation. Therefore, there are two strategies for bioenergy production for Tribonema sp., short-term cultivation for ethanol fermentation and long-term cultivation for biodiesel production, due to the dominant components are carbohydrate and lipid, respectively.
3.4. Triacylglycerols (TAGs) accumulation to different levels of nitrogen in Tribonema Many investigations focused on the impact of nitrogen starvation on total lipid accumulation whereas only a few reports are available about the impact of nitrogen limitation on TAG accumulation. Triacylglycerols (TAGs) are stored in specialized cytosolic oil bodies and function as energy reserve, and more desirable than phospholipids and glycolipids for biodiesel production as they have a higher FA (fatty acid) content and no phosphate (Mata et al., 2010; Miao and Wu, 2006). TAGs contents of Tribonema in different nitrogen levels medium at the end of cultivation were analyzed using TLC–FID (Table 1). In contrast to the impact on the biomass and total lipid content, slight differences were observed in the TAG contents (% of lipid) in microalgae cultured with different levels of nitrogen. TAG was the majority composition in total lipid with all treatments, accounted for ca. 82.77–87.5% (of total lipid). However, significant distinctions existed among the TAG contents in dry weight, as the lipid contents of dry weight linearly decreased from high levels to low levels of nitrogen. The final TAG
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Fig. 3. Dynamics of cellular components, protein (A), lipid (B), soluble sugar (C) and insoluble sugar (D) in Tribonema, in response to 0N, 0.05N, 0.1N and 1N-BG11 medium.
Table 1 TAG contents in microalgae with different levels of nitrogen supply. Nitrogen concentration
TAG (% of lipid) TAG (% of dry weight)
0N-BG11
0.05NBG11
0.1NBG11
1N-BG11
82.77 ± 1.2 37.74 ± 0.3
83.96 ± 0.9 40.45 ± 2.4
86.31 ± 1.7 45.13 ± 1.1
87.5 ± 4.3 49.89 ± 2.3
contents of dry weight were 37.74%, 40.45%, 45.13% and 49.89%, respectively, in response to 0N, 0.05N, 0.1N and 1N-BG11. To our knowledge, in general, a common practice to stimulate TAG accumulation in microalgae is to cultivate microalgae longer to produce a natural nitrogen stress environments by the gradually used up of nitrogen as this way is cost effective and easy to manipulate (Brennan and Qwende, 2010). The total fatty acid content has a minor decrease upon nitrogen starvation,which agreed with those by Dunaliella tertiolecta (Guido et al., 2013), though the reasons are unknown. 3.5. Fatty acid profiles of Tribonema under different nitrogen levels In addition to TAGs content, fatty acid profile of microalgae is also an important characteristic as it ultimately affects the quality of the biodiesel product (Gouveia and Oliveira, 2009; Knothe, 2005). Fatty acid profiles of Tribonema were determined at the end of cultivation (day 15) under all nitrogen levels (Fig. 4). Among all conditions, palmitoleic acid (C16:1) was always the dominant fatty acid, constituting up to ca. 49.09–59.37% of total fatty acids in Tribonema, while oleic acid was in majority in green algae (Griffiths et al., 2011). The second main composition was palmitic acid (C16:0). However, the oleic acid (C18:1) in Tribonema only accounted for 3.94%, 3.81%, 3.7%, and 2.49%, respectively, in response to 0N, 0.05N, 0.1N and 1N-BG11 medium. Furthermore, eicosapentaenoic (C20:5) existed among all treatments, the contents were from 2.58% to 2.93% (% of total fatty acid). In total, fatty acid compositions upon all levels of nitrogen were very similar, though 8.4% of myristic acid (C14:0) and 49.1% of
Fig. 4. Fatty acid profiles of Tribonema with different levels of nitrogen supply.
palmitoleic (C16:1) were observed in nitrogen-free medium (0N-BG11) while those of 5.16% and 56.66%, respectively in nitrogen-replete medium (1N-BG11). The result meant that different nitrogen levels did not cause fundamental shifts in fatty acid compositions of lipid from filamentous microalgae Tribonema.
4. Conclusion Filamentous oleaginous microalgae Tribonema is promising feedstock for biofuel production. However, it had different biochemical response compared with general unicellular oleaginous microalgae. There is no starch synthesis pathway in the Tribonema cells. Carbohydrate (cellulose) was the dominated component in mid-term of the cultivation, while lipid was the main component in late stage of cultivation. In addition, total protein and TAG(s) contents (% of dry weight) decreased in response to the increment of carbohydrate (mainly cellulose) in Tribonema cells under nitrogen starvation conditions. Therefore, further research on the
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correlative relationship between synthesis of carbohydrate and lipid in Tribonema is recommended.
Acknowledgements This work was supported by Qibebt (CAS)-Boeing Joint Research Laboratory for Sustainable Aviation Biofuel, the Director Innovation Foundation of Qibebt (CAS) and Science and the technology development plan of Shandong Province (2013GGF01008).
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