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Combined effects of nitrogen levels and Daphnia culture filtrate on colony size of Scenedesmus obliquus
Algal Research 9 (2015) 94–98
Contents lists available at ScienceDirect
Algal Research journal homepage: www.elsevier.com/locate/algal
Short communication
Combined effects of nitrogen levels and Daphnia culture filtrate on colony size of Scenedesmus obliquus Xuexia Zhu, Jingwen Yang, Nuo Xu, Ge Chen, Zhou Yang ⁎ Jiangsu Key Laboratory for Biodiversity and Biotechnology, School of Biological Sciences, Nanjing Normal University, Nanjing 210023, China
a r t i c l e
i n f o
Article history: Received 20 November 2014 Received in revised form 2 March 2015 Accepted 4 March 2015 Available online xxxx Keywords: Colony formation Daphnia culture filtrate Microalgae Morphological change Polysaccharides
a b s t r a c t Increased colony size facilitates the harvest of Scenedesmus cells from culture media; as such, factors that stimulate colony enlargement for production should be identified. This study aimed to investigate the combined effects of nitrogen levels and Daphnia culture filtrate on the colony size of Scenedesmus obliquus. Results showed that the colony formation of S. obliquus induced by Daphnia culture filtrate was promoted under low nitrogen levels. Colony size increased by approximately 50%–100%, which suggested that low nitrogen and zooplankton infochemicals synergistically affected the colony formation of S. obliquus. Furthermore, increased colony size and high settling velocities enhanced the suitability of populations for centrifugation and filtration; thus, these populations could be helpful for harvest. According to the results, addition of Daphnia culture filtrate at 3 days before S. obliquus was harvested could be useful. Therefore, the proposed method could be applied to facilitate efficient harvest of Scenedesmus biomass. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Microalgal cultivation has been considered as a feasible method to produce valuable chemical compounds and biodiesel [1]. Efficient biomass harvesting is also a key step for the mass production of valuable substances from microalgae [2,3]. Microalgae may be harvested commercially using centrifugation, filtration, and flocculation, either used individually or in combination [3]. Filtration is suitable for colonyforming microalgae, such as Scenedesmus, which has been developed and cultivated as a very important alga for biodiesel feedstock because this organism can grow in various types of wastewater and also exhibits high biomass, lipid, and carbohydrate contents [4]. However, separation and recovery of Scenedesmus from culture media have been considered as a critical step in algal biomass production because of small size and retaining stability in a dispersed state [5]. As such, efficient and lowcost downstream processes should involve methods in which colony size of Scenedesmus cells is increased to harvest these cells from culture media. Therefore, factors that stimulate this increase in aggregate size during production should be identified. From this view, it is essential to develop ecological technologies to change the morphological trait of Scenedesmus. Potential grazing pressure from herbivorous zooplankton may be a feasible way to increase the colony size of Scenedesmus. Scenedesmus, usually dominated by unicells and paired cells, can be induced to form ⁎ Corresponding author at: Jiangsu Key Laboratory for Biodiversity and Biotechnology, School of Biological Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210023, China. E-mail address: [email protected] (Z. Yang).
http://dx.doi.org/10.1016/j.algal.2015.03.002 2211-9264/© 2015 Elsevier B.V. All rights reserved.
large colonies by Daphnia culture filtrates [6–8]. Furthermore, extracellular polysaccharides increase the stickiness of the cell surface and contribute to cell aggregation in algal species [9–11]. Extracellular polysaccharides can be stimulated under lower nitrogen conditions [12,13]. Considering these observations, we hypothesized that colony formation of Scenedesmus may be enhanced by synergistic effects between low nitrogen and zooplankton infochemicals. To test this hypothesis, we used Scenedesmus obliquus and examined its morphological response to the combination of nitrogen levels and Daphnia culture filtrate. If our results support our hypothesis, this colony-enlarging approach likely facilitates the harvest of Scenedesmus from culture media for downstream processing. 2. Materials and methods 2.1. Test organisms and Daphnia culture filtrate preparation S. obliquus strain FACHB-416 was axenically maintained in liquid BG11 medium at 25 °C in a 12 h:12 h light:dark cycle at 40 μmol photons m−2 s−1. The zooplankton grazer Daphnia magna was cultured in beakers and fed with S. obliquus (5 × 105 cells mL−1) under the same conditions as described above. Daphnia culture filtrate was then obtained as previously described [14,15]. In brief, D. magna cultures were collected, washed to remove surface residual medium, and allowed to fast in water for at least 12 h; afterward, D. magna cultures were incubated at a density of 200 ind L− 1 and fed with sufficient S. obliquus. After 24 h, D. magna cultures were removed; water from these cultures was filtered through a 0.10 μm membrane filter (Millipore Corporation, USA) and test water containing Daphnia infochemicals was produced.
X. Zhu et al. / Algal Research 9 (2015) 94–98
2.2. Experimental design To minimize intracellular nutrient storage and nitrogen in the medium, S. obliquus cultures (50 mL) in exponential growth were harvested by centrifugation (6000 × g for 15 min) and then re-suspended in nitrogen-free BG-11 medium for two days, which was repeated twice in four days. After four days, the cells were collected by centrifugation, and the pellets were inoculated into modified BG-11 medium with different nitrogen contents (32, 16, 8, 4, and 2 mg L−1) in 250 mL flasks. The corresponding average measured nitrogen contents were 33.05, 14.69, 7.75, 3.51, and 2.09 mg L−1. The initial algal concentration in the treatment groups and the control group was approximately 5.5 × 104 cells mL−1. Each flask contained 135 mL of S. obliquus suspension and either 15 mL of additional Daphnia culture filtrate or 15 mL of Scenedesmus culture filtrate. The experiments were run in three replicates for seven days under the conditions as described in Section 2.1. The cultures were shaken manually twice a day to maintain culture homogeneity. 2.3. Measurement of growth rate and colony size of S. obliquus The number of cells per particle of S. obliquus was measured in all of the flasks during the experiment to determine the combined effects of nitrogen levels and Daphnia culture filtrate on morphological changes in S. obliquus. Samples (2 mL) were collected daily and fixed in Lugol's solution (2%). Abundance was determined using a hemocytometer (Tianlong XB-K-25; Jiangsu, China) under a microscope (Olympus 6V20WHAL; Tokyo, Japan). The numbers of cells per particle were calculated by counting at least 600 particles (i.e., unicells, two-celled, four-celled, eight-celled, and the rest) under a light microscope. Growth rate was determined as the slope of logarithm abundance versus time from the samples collected daily [11]. 2.4. Statistical analyses Data were presented as means ± 1 SE. Growth and cells per particle were analyzed by two-way ANOVA. Statistical analyses were performed in SigmaPlot 11.0. 3. Results and discussion S. obliquus grew well under all nitrogen levels in this study. Two-way ANOVA results indicated that adding Daphnia culture filtrate did not affect the growth of S. obliquus; this result is consistent with that described in other studies [8,14,15]. Although nitrogen level significantly affected the growth rate of S. obliquus (Table 1), the differential degree was very low; in particular, growth rate only decreased by approximately 6% from 0.54 d−1 at 32 mg L− 1 N to 0.48 d− 1 at 2 mg L− 1 N (Fig. 1). In the treatments with added Daphnia culture filtrate, the proportion of eight-celled colonies increased rapidly and reached the peak on day 3
Table 1 Summary of two-way ANOVA of the effects of nitrogen level and Daphnia culture filtrate on the growth rate of S. obliquus. Source of variation
DF SS
Nitrogen level 4 Daphnia culture filtrate 1 Nitrogen level × Daphnia culture filtrate 4
MS
F
P
0.0188 0.00471 3.303 0.031 0.000881 0.000881 0.618 0.441 0.000337 0.0000842 0.0591 0.993
0.6
SF DF
0.5
Growth rate (d-1)
For the control filtrate, a culture of S. obliquus without Daphnia was subjected to the same procedures as producing Daphnia culture filtrate. Nitrate and phosphate concentrations in Daphnia culture filtrate and Scenedesmus culture filtrate were adjusted to the same levels before these filtrates were used in the experiments.
95
0.4 0.3 0.2 0.1 0.0 32 16 8 4 2 -1 Nitrogen levels (mg L )
Fig. 1. Growth rates of S. obliquus populations incubated under different conditions during the 7-day experiment. Vertical lines represent one SE. SF: Scenedesmus culture filtrate; DF: Daphnia culture filtrate.
(Fig. 2); this peak was evidently higher than that of the cultures with added Scenedesmus culture filtrate. This observation was in line with that described in other studies [6–8,14,15]. The effect of nitrogen level was also determined in this study. The proportions of cells in colonies at low nitrogen levels (2 mg L−1 N) were higher than those at high nitrogen levels (4–32 mg L−1 N) after day 3. Rapid morphological responses of S. obliquus in the experiments were also indicated by the mean number of cells per particle (Fig. 3). In general, colony size increased by approximately 50%–100% in the cultures with added Daphnia culture filtrate. Two-way ANOVA results showed that low nitrogen levels and Daphnia culture filtrate significantly affected the mean numbers of cells per particle of S. obliquus; significant synergistic effects were detected between nitrogen levels and Daphnia culture filtrate on colony formation at the end of the experiments (Table 2). The colony formation of S. obliquus in this study was likely a consequence of increased polysaccharide content in cells, as demonstrated in other studies [16,17]. Moreover, nitrate stress can possibly induce an increase in carbohydrate content [18]; excess carbohydrate is preferentially channeled by algal cells to synthesize high amounts of products, such as polysaccharides [19], thereby contributing to cell aggregation. Therefore, colony formation of S. obliquus induced by Daphnia culture filtrate was promoted under low nitrogen levels probably because high amounts of polysaccharides were produced by S. obliquus under low nitrogen conditions. This result suggested that nitrogen level and Daphnia culture filtrate elicited a combined effect on the colony size of S. obliquus; this finding is similar to the phenomenon observed in Microcystis [20]. Furthermore, large colonial populations exhibit higher settling velocities than unicellular populations [15]. Both increased colony size and higher settling velocities enhance the suitability of populations for centrifugation and filtration; thus, these factors could be considered to harvest S. obliquus. Our results also showed that Daphnia culture filtrate should be added 3 days before S. obliquus was harvested from culture media. Furthermore, from the view of increasing carbohydrate content and facilitating harvest of Scenedesmus, such colonyenlarging approach is really an effective method to satisfy both sides.
4. Conclusions In summary, S. obliquus can grow well in different nitrogen levels. Colony formation of S. obliquus induced by Daphnia culture filtrate can be promoted under low nitrogen levels (2 mg L−1 N); this result suggested that nitrogen level and Daphnia culture filtrate elicited a
X. Zhu et al. / Algal Research 9 (2015) 94–98
Proportion of population
Proportion of population
Proportion of population
Proportion of population
Proportion of population
96
1.0
1.0
32 mg L-1 N + SF
unicell 2-cell 4-cell 8-cell rest
0.8 0.6
0.6
0.4
0.4
0.2
0.2
0.0 1.0
0.0 1.0
16 mg L-1 N + SF
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
1.0
1.0
0.8
8 mg L-1 N + SF
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0 1.0
0.0 1.0
4 mg L-1 N + SF
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0 1.0
0.0 1.0
2 mg L-1 N + SF
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0 1
2
3 4 5 Time (d)
6
7
32 mg L-1 N + DF
0.8
16 mg L-1 N + DF
8 mg L-1 N + DF
2D Graph 6
4 mg L-1 N + DF
2 mg L-1 N + DF
1
2
3 4 5 Time (d)
6
7
Fig. 2. Proportions of unicells and two-, four-, and eight-celled colonies in S. obliquus populations incubated under different conditions during the 7-day experiment. The “rest” group represents three-, five-, six-, seven-, and multi-celled colonies. SF: Scenedesmus culture filtrate; DF: Daphnia culture filtrate.
combined effect on the colony size of S. obliquus. Therefore, the proposed method could be applied to facilitate harvest of Scenedesmus cells.
Conflict of interest The authors declare that there is no conflict of interest.
Author's contributions Acknowledgments All authors contributed to the conception and design of the study, or acquisition of data, or analysis and interpretation of data; drafting the article or revising it critically for important intellectual content final, and all authors approve of the version to be submitted. Zhou Yang took responsibility for the integrity of the work as a whole, from inception to finished article.
This study was supported by the National Natural Science Foundation of China (31270504, 31470508) and the Priority Academic Program Development of Jiangsu Higher Education Institutions. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
X. Zhu et al. / Algal Research 9 (2015) 94–98
Cells per particle
6
6 32 mg L-1 N + SF 32 mg L-1 N + DF
5
4
3
3
2
2
1
1 6 8 mg L-1 N + SF 8 mg L-1 N + DF
5
16 mg L-1 N + SF 16 mg L-1 N + DF
5
4
6
Cells per particle
97
4 mg L-1 N + SF 4 mg L-1 N + DF
5
4
4
3
3
2
2 1
1
6
2 mg L-1 N + SF 2 mg L-1 N + DF
1
2
3
4 5 Time (d)
6
7
Cells per particle
5 4 3 2 1 1
2
3
4 5 Time (d)
6
7
Fig. 3. Changes in the mean number of cells per particle of S. obliquus incubated under different conditions during the 7-day experiment. Vertical lines represent one SE. SF: Scenedesmus culture filtrate; DF: Daphnia culture filtrate.
Table 2 Summary of two-way ANOVA of the effects of nitrogen level and Daphnia culture filtrate on the number of cells per particle of S. obliquus. Time
Source of variation
DF
SS
MS
F
P
Day 2
Daphnia culture filtrate Nitrogen level Daphnia culture filtrate × nitrogen level Daphnia culture filtrate Nitrogen level Daphnia culture filtrate × nitrogen level Daphnia culture filtrate Nitrogen level Daphnia culture filtrate × nitrogen level Daphnia culture filtrate Nitrogen level Daphnia culture filtrate × nitrogen level Daphnia culture filtrate Nitrogen level Daphnia culture filtrate × nitrogen level Daphnia culture filtrate Nitrogen level Daphnia culture filtrate × nitrogen level
1 4 4 1 4 4 1 4 4 1 4 4 1 4 4 1 4 4
12.023 7.428 3.675 20.237 1.889 0.704 11.186 4.380 1.132 8.117 8.745 0.788 5.232 9.171 2.078 5.973 3.715 3.106
12.023 1.857 0.919 20.237 0.472 0.176 11.186 1.095 0.283 8.117 2.186 0.197 5.232 2.293 0.519 5.973 0.929 0.776
50.173 7.749 3.834 73.845 1.723 0.642 49.807 4.876 1.260 33.299 8.969 0.808 53.104 23.273 5.273 22.729 3.534 2.954
b0.001 b0.001 0.018 b0.001 0.184 0.639 b0.001 0.007 0.318 b0.001 b0.001 0.535 b0.001 b0.001 0.005 b0.001 0.025 0.045
Day 3
Day 4
Day 5
Day 6
Day 7
98
X. Zhu et al. / Algal Research 9 (2015) 94–98
References [1] O. Pulz, W. Gross, Valuable products from biotechnology of microalgae, Appl. Microbiol. Biotechnol. 65 (6) (2004) 635–648. [2] Y.-L. Cheng, Y.-C. Juang, G.-Y. Liao, P.-W. Tsai, S.-H. Ho, K.-L. Yeh, et al., Harvesting of Scenedesmus obliquus FSP-3 using dispersed ozone flotation, Bioresour. Technol. 102 (1) (2011) 82–87. [3] A.K. Lee, D.M. Lewis, P.J. Ashman, Microbial flocculation, a potentially low-cost harvesting technique for marine microalgae for the production of biodiesel, J. Appl. Phycol. 21 (5) (2009) 559–567. [4] T.M. Mata, A.C. Melo, M. Simões, N.S. Caetano, Parametric study of a brewery effluent treatment by microalgae Scenedesmus obliquus, Bioresour. Technol. 107 (2012) 151–158. [5] L. Chen, C. Wang, W. Wang, J. Wei, Optimal conditions of different flocculation methods for harvesting Scenedesmus sp. cultivated in an open-pond system, Bioresour. Technol. 133 (2013) 9–15. [6] M. Lürling, E. Van Donk, Morphological changes in Scenedesmus induced by infochemicals released in situ from zooplankton grazers, Limnol. Oceanogr. 42 (4) (1997) 783–788. [7] Z. Yang, F.X. Kong, X.L. Shi, H.S. Cao, Differences in response to rotifer Brachionus urceus culture media filtrate between Scenedesmus obliquus and Microcystis aeruginosa, J. Freshw. Ecol. 21 (2) (2006) 209–214. [8] E. Van Donk, A. Ianora, M. Vos, Induced defences in marine and freshwater phytoplankton: a review, Hydrobiologia 668 (1) (2011) 3–19. [9] D. Thornton, Diatom aggregation in the sea: mechanisms and ecological implications, Eur. J. Phycol. 37 (2) (2002) 149–161. [10] Z. Yang, F.X. Kong, X.L. Shi, M. Zhang, P. Xing, H.S. Cao, Changes in the morphology and polysaccharide content of Microcystis aeruginosa (cyanobacteria) during flagellate grazing, J. Phycol. 44 (3) (2008) 716–720. [11] Z. Yang, Y. Liu, J. Ge, W. Wang, Y.F. Chen, D.J.S. Montagnes, Aggregate formation and polysaccharide content of Chlorella pyrenoidosa Chick (Chlorophyta) in response to simulated nutrient stress, Bioresour. Technol. 101 (21) (2010) 8336–8341.
[12] E. Granum, S. Kirkvold, S.M. Myklestad, Cellular and extracellular production of carbohydrates and amino acids by the marine diatom Skeletonema costatum: diel variations and effects of N depletion, Mar. Ecol. Prog. Ser. 242 (2002) 83–94. [13] E. Magaletti, R. Urbani, P. Sist, C.R. Ferrari, A.M. Cicero, Abundance and chemical characterization of extracellular carbohydrates released by the marine diatom Cylindrotheca fusiformis under N-and P-limitation, Eur. J. Phycol. 39 (2) (2004) 133–142. [14] X.X. Zhu, H.H. Nan, L. Zhang, C. Zhu, J. Liu, Z. Yang, Does microcystin disrupt the induced effect of Daphnia kairomone on colony formation in Scenedesmus? Biochem. Syst. Ecol. 57 (2014) 169–174. [15] M. Lürling, E. Van Donk, Grazer-induced colony formation in Scenedesmus: are there costs to being colonial? Oikos 88 (1) (2000) 111–118. [16] Y. Liu, W. Wang, M. Zhang, P. Xing, Z. Yang, PSII-efficiency, polysaccharide production, and phenotypic plasticity of Scenedesmus obliquus in response to changes in metabolic carbon flux, Biochem. Syst. Ecol. 38 (3) (2010) 292–299. [17] Z. Yang, F.X. Kong, X.L. Shi, P. Xing, M. Zhang, Effects of Daphnia-associated infochemicals on the morphology, polysaccharides content and PSII-efficiency in Scenedesmus obliquus, Int. Rev. Hydrobiol. 92 (6) (2007) 618–625. [18] I. Pancha, K. Chokshi, B. George, T. Ghosh, C. Paliwal, R. Maurya, et al., Nitrogen stress triggered biochemical and morphological changes in the microalgae Scenedesmus sp. CCNM 1077, Bioresour. Technol. 156 (2014) 146–154. [19] R. De Philippis, C. Sili, M. Vincenzini, Response of an exopolysaccharide-producing heterocystous cyanobacterium to changes in metabolic carbon flux, J. Appl. Phycol. 8 (4–5) (1996) 275–281. [20] W. Wang, Y. Liu, Z. Yang, Combined effects of nitrogen content in media and Ochromonas sp. grazing on colony formation of cultured Microcystis aeruginosa, J. Limnol. 69 (2) (2010) 193–198.
Contents lists available at ScienceDirect
Algal Research journal homepage: www.elsevier.com/locate/algal
Short communication
Combined effects of nitrogen levels and Daphnia culture filtrate on colony size of Scenedesmus obliquus Xuexia Zhu, Jingwen Yang, Nuo Xu, Ge Chen, Zhou Yang ⁎ Jiangsu Key Laboratory for Biodiversity and Biotechnology, School of Biological Sciences, Nanjing Normal University, Nanjing 210023, China
a r t i c l e
i n f o
Article history: Received 20 November 2014 Received in revised form 2 March 2015 Accepted 4 March 2015 Available online xxxx Keywords: Colony formation Daphnia culture filtrate Microalgae Morphological change Polysaccharides
a b s t r a c t Increased colony size facilitates the harvest of Scenedesmus cells from culture media; as such, factors that stimulate colony enlargement for production should be identified. This study aimed to investigate the combined effects of nitrogen levels and Daphnia culture filtrate on the colony size of Scenedesmus obliquus. Results showed that the colony formation of S. obliquus induced by Daphnia culture filtrate was promoted under low nitrogen levels. Colony size increased by approximately 50%–100%, which suggested that low nitrogen and zooplankton infochemicals synergistically affected the colony formation of S. obliquus. Furthermore, increased colony size and high settling velocities enhanced the suitability of populations for centrifugation and filtration; thus, these populations could be helpful for harvest. According to the results, addition of Daphnia culture filtrate at 3 days before S. obliquus was harvested could be useful. Therefore, the proposed method could be applied to facilitate efficient harvest of Scenedesmus biomass. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Microalgal cultivation has been considered as a feasible method to produce valuable chemical compounds and biodiesel [1]. Efficient biomass harvesting is also a key step for the mass production of valuable substances from microalgae [2,3]. Microalgae may be harvested commercially using centrifugation, filtration, and flocculation, either used individually or in combination [3]. Filtration is suitable for colonyforming microalgae, such as Scenedesmus, which has been developed and cultivated as a very important alga for biodiesel feedstock because this organism can grow in various types of wastewater and also exhibits high biomass, lipid, and carbohydrate contents [4]. However, separation and recovery of Scenedesmus from culture media have been considered as a critical step in algal biomass production because of small size and retaining stability in a dispersed state [5]. As such, efficient and lowcost downstream processes should involve methods in which colony size of Scenedesmus cells is increased to harvest these cells from culture media. Therefore, factors that stimulate this increase in aggregate size during production should be identified. From this view, it is essential to develop ecological technologies to change the morphological trait of Scenedesmus. Potential grazing pressure from herbivorous zooplankton may be a feasible way to increase the colony size of Scenedesmus. Scenedesmus, usually dominated by unicells and paired cells, can be induced to form ⁎ Corresponding author at: Jiangsu Key Laboratory for Biodiversity and Biotechnology, School of Biological Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210023, China. E-mail address: [email protected] (Z. Yang).
http://dx.doi.org/10.1016/j.algal.2015.03.002 2211-9264/© 2015 Elsevier B.V. All rights reserved.
large colonies by Daphnia culture filtrates [6–8]. Furthermore, extracellular polysaccharides increase the stickiness of the cell surface and contribute to cell aggregation in algal species [9–11]. Extracellular polysaccharides can be stimulated under lower nitrogen conditions [12,13]. Considering these observations, we hypothesized that colony formation of Scenedesmus may be enhanced by synergistic effects between low nitrogen and zooplankton infochemicals. To test this hypothesis, we used Scenedesmus obliquus and examined its morphological response to the combination of nitrogen levels and Daphnia culture filtrate. If our results support our hypothesis, this colony-enlarging approach likely facilitates the harvest of Scenedesmus from culture media for downstream processing. 2. Materials and methods 2.1. Test organisms and Daphnia culture filtrate preparation S. obliquus strain FACHB-416 was axenically maintained in liquid BG11 medium at 25 °C in a 12 h:12 h light:dark cycle at 40 μmol photons m−2 s−1. The zooplankton grazer Daphnia magna was cultured in beakers and fed with S. obliquus (5 × 105 cells mL−1) under the same conditions as described above. Daphnia culture filtrate was then obtained as previously described [14,15]. In brief, D. magna cultures were collected, washed to remove surface residual medium, and allowed to fast in water for at least 12 h; afterward, D. magna cultures were incubated at a density of 200 ind L− 1 and fed with sufficient S. obliquus. After 24 h, D. magna cultures were removed; water from these cultures was filtered through a 0.10 μm membrane filter (Millipore Corporation, USA) and test water containing Daphnia infochemicals was produced.
X. Zhu et al. / Algal Research 9 (2015) 94–98
2.2. Experimental design To minimize intracellular nutrient storage and nitrogen in the medium, S. obliquus cultures (50 mL) in exponential growth were harvested by centrifugation (6000 × g for 15 min) and then re-suspended in nitrogen-free BG-11 medium for two days, which was repeated twice in four days. After four days, the cells were collected by centrifugation, and the pellets were inoculated into modified BG-11 medium with different nitrogen contents (32, 16, 8, 4, and 2 mg L−1) in 250 mL flasks. The corresponding average measured nitrogen contents were 33.05, 14.69, 7.75, 3.51, and 2.09 mg L−1. The initial algal concentration in the treatment groups and the control group was approximately 5.5 × 104 cells mL−1. Each flask contained 135 mL of S. obliquus suspension and either 15 mL of additional Daphnia culture filtrate or 15 mL of Scenedesmus culture filtrate. The experiments were run in three replicates for seven days under the conditions as described in Section 2.1. The cultures were shaken manually twice a day to maintain culture homogeneity. 2.3. Measurement of growth rate and colony size of S. obliquus The number of cells per particle of S. obliquus was measured in all of the flasks during the experiment to determine the combined effects of nitrogen levels and Daphnia culture filtrate on morphological changes in S. obliquus. Samples (2 mL) were collected daily and fixed in Lugol's solution (2%). Abundance was determined using a hemocytometer (Tianlong XB-K-25; Jiangsu, China) under a microscope (Olympus 6V20WHAL; Tokyo, Japan). The numbers of cells per particle were calculated by counting at least 600 particles (i.e., unicells, two-celled, four-celled, eight-celled, and the rest) under a light microscope. Growth rate was determined as the slope of logarithm abundance versus time from the samples collected daily [11]. 2.4. Statistical analyses Data were presented as means ± 1 SE. Growth and cells per particle were analyzed by two-way ANOVA. Statistical analyses were performed in SigmaPlot 11.0. 3. Results and discussion S. obliquus grew well under all nitrogen levels in this study. Two-way ANOVA results indicated that adding Daphnia culture filtrate did not affect the growth of S. obliquus; this result is consistent with that described in other studies [8,14,15]. Although nitrogen level significantly affected the growth rate of S. obliquus (Table 1), the differential degree was very low; in particular, growth rate only decreased by approximately 6% from 0.54 d−1 at 32 mg L− 1 N to 0.48 d− 1 at 2 mg L− 1 N (Fig. 1). In the treatments with added Daphnia culture filtrate, the proportion of eight-celled colonies increased rapidly and reached the peak on day 3
Table 1 Summary of two-way ANOVA of the effects of nitrogen level and Daphnia culture filtrate on the growth rate of S. obliquus. Source of variation
DF SS
Nitrogen level 4 Daphnia culture filtrate 1 Nitrogen level × Daphnia culture filtrate 4
MS
F
P
0.0188 0.00471 3.303 0.031 0.000881 0.000881 0.618 0.441 0.000337 0.0000842 0.0591 0.993
0.6
SF DF
0.5
Growth rate (d-1)
For the control filtrate, a culture of S. obliquus without Daphnia was subjected to the same procedures as producing Daphnia culture filtrate. Nitrate and phosphate concentrations in Daphnia culture filtrate and Scenedesmus culture filtrate were adjusted to the same levels before these filtrates were used in the experiments.
95
0.4 0.3 0.2 0.1 0.0 32 16 8 4 2 -1 Nitrogen levels (mg L )
Fig. 1. Growth rates of S. obliquus populations incubated under different conditions during the 7-day experiment. Vertical lines represent one SE. SF: Scenedesmus culture filtrate; DF: Daphnia culture filtrate.
(Fig. 2); this peak was evidently higher than that of the cultures with added Scenedesmus culture filtrate. This observation was in line with that described in other studies [6–8,14,15]. The effect of nitrogen level was also determined in this study. The proportions of cells in colonies at low nitrogen levels (2 mg L−1 N) were higher than those at high nitrogen levels (4–32 mg L−1 N) after day 3. Rapid morphological responses of S. obliquus in the experiments were also indicated by the mean number of cells per particle (Fig. 3). In general, colony size increased by approximately 50%–100% in the cultures with added Daphnia culture filtrate. Two-way ANOVA results showed that low nitrogen levels and Daphnia culture filtrate significantly affected the mean numbers of cells per particle of S. obliquus; significant synergistic effects were detected between nitrogen levels and Daphnia culture filtrate on colony formation at the end of the experiments (Table 2). The colony formation of S. obliquus in this study was likely a consequence of increased polysaccharide content in cells, as demonstrated in other studies [16,17]. Moreover, nitrate stress can possibly induce an increase in carbohydrate content [18]; excess carbohydrate is preferentially channeled by algal cells to synthesize high amounts of products, such as polysaccharides [19], thereby contributing to cell aggregation. Therefore, colony formation of S. obliquus induced by Daphnia culture filtrate was promoted under low nitrogen levels probably because high amounts of polysaccharides were produced by S. obliquus under low nitrogen conditions. This result suggested that nitrogen level and Daphnia culture filtrate elicited a combined effect on the colony size of S. obliquus; this finding is similar to the phenomenon observed in Microcystis [20]. Furthermore, large colonial populations exhibit higher settling velocities than unicellular populations [15]. Both increased colony size and higher settling velocities enhance the suitability of populations for centrifugation and filtration; thus, these factors could be considered to harvest S. obliquus. Our results also showed that Daphnia culture filtrate should be added 3 days before S. obliquus was harvested from culture media. Furthermore, from the view of increasing carbohydrate content and facilitating harvest of Scenedesmus, such colonyenlarging approach is really an effective method to satisfy both sides.
4. Conclusions In summary, S. obliquus can grow well in different nitrogen levels. Colony formation of S. obliquus induced by Daphnia culture filtrate can be promoted under low nitrogen levels (2 mg L−1 N); this result suggested that nitrogen level and Daphnia culture filtrate elicited a
X. Zhu et al. / Algal Research 9 (2015) 94–98
Proportion of population
Proportion of population
Proportion of population
Proportion of population
Proportion of population
96
1.0
1.0
32 mg L-1 N + SF
unicell 2-cell 4-cell 8-cell rest
0.8 0.6
0.6
0.4
0.4
0.2
0.2
0.0 1.0
0.0 1.0
16 mg L-1 N + SF
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
1.0
1.0
0.8
8 mg L-1 N + SF
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0 1.0
0.0 1.0
4 mg L-1 N + SF
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0 1.0
0.0 1.0
2 mg L-1 N + SF
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0 1
2
3 4 5 Time (d)
6
7
32 mg L-1 N + DF
0.8
16 mg L-1 N + DF
8 mg L-1 N + DF
2D Graph 6
4 mg L-1 N + DF
2 mg L-1 N + DF
1
2
3 4 5 Time (d)
6
7
Fig. 2. Proportions of unicells and two-, four-, and eight-celled colonies in S. obliquus populations incubated under different conditions during the 7-day experiment. The “rest” group represents three-, five-, six-, seven-, and multi-celled colonies. SF: Scenedesmus culture filtrate; DF: Daphnia culture filtrate.
combined effect on the colony size of S. obliquus. Therefore, the proposed method could be applied to facilitate harvest of Scenedesmus cells.
Conflict of interest The authors declare that there is no conflict of interest.
Author's contributions Acknowledgments All authors contributed to the conception and design of the study, or acquisition of data, or analysis and interpretation of data; drafting the article or revising it critically for important intellectual content final, and all authors approve of the version to be submitted. Zhou Yang took responsibility for the integrity of the work as a whole, from inception to finished article.
This study was supported by the National Natural Science Foundation of China (31270504, 31470508) and the Priority Academic Program Development of Jiangsu Higher Education Institutions. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
X. Zhu et al. / Algal Research 9 (2015) 94–98
Cells per particle
6
6 32 mg L-1 N + SF 32 mg L-1 N + DF
5
4
3
3
2
2
1
1 6 8 mg L-1 N + SF 8 mg L-1 N + DF
5
16 mg L-1 N + SF 16 mg L-1 N + DF
5
4
6
Cells per particle
97
4 mg L-1 N + SF 4 mg L-1 N + DF
5
4
4
3
3
2
2 1
1
6
2 mg L-1 N + SF 2 mg L-1 N + DF
1
2
3
4 5 Time (d)
6
7
Cells per particle
5 4 3 2 1 1
2
3
4 5 Time (d)
6
7
Fig. 3. Changes in the mean number of cells per particle of S. obliquus incubated under different conditions during the 7-day experiment. Vertical lines represent one SE. SF: Scenedesmus culture filtrate; DF: Daphnia culture filtrate.
Table 2 Summary of two-way ANOVA of the effects of nitrogen level and Daphnia culture filtrate on the number of cells per particle of S. obliquus. Time
Source of variation
DF
SS
MS
F
P
Day 2
Daphnia culture filtrate Nitrogen level Daphnia culture filtrate × nitrogen level Daphnia culture filtrate Nitrogen level Daphnia culture filtrate × nitrogen level Daphnia culture filtrate Nitrogen level Daphnia culture filtrate × nitrogen level Daphnia culture filtrate Nitrogen level Daphnia culture filtrate × nitrogen level Daphnia culture filtrate Nitrogen level Daphnia culture filtrate × nitrogen level Daphnia culture filtrate Nitrogen level Daphnia culture filtrate × nitrogen level
1 4 4 1 4 4 1 4 4 1 4 4 1 4 4 1 4 4
12.023 7.428 3.675 20.237 1.889 0.704 11.186 4.380 1.132 8.117 8.745 0.788 5.232 9.171 2.078 5.973 3.715 3.106
12.023 1.857 0.919 20.237 0.472 0.176 11.186 1.095 0.283 8.117 2.186 0.197 5.232 2.293 0.519 5.973 0.929 0.776
50.173 7.749 3.834 73.845 1.723 0.642 49.807 4.876 1.260 33.299 8.969 0.808 53.104 23.273 5.273 22.729 3.534 2.954
b0.001 b0.001 0.018 b0.001 0.184 0.639 b0.001 0.007 0.318 b0.001 b0.001 0.535 b0.001 b0.001 0.005 b0.001 0.025 0.045
Day 3
Day 4
Day 5
Day 6
Day 7
98
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