Appl Biochem Biotechnol DOI 10.1007/s12010-014-1283-6
Nitrogen Starvation for Lipid Accumulation in the Microalga Species Desmodesmus sp. L. F. Rios & B. C. Klein & L. F. Luz Jr. & R. Maciel Filho & M. R. Wolf Maciel
Received: 6 May 2014 / Accepted: 2 October 2014 # Springer Science+Business Media New York 2014
Abstract Recently, to obtain lipids from microalgae has been the object of extensive research, since it is viewed as a promising feedstock for biodiesel production, especially when compared with crops such as soybean and sunflower, in terms of theoretical performance. The reduction of nutrient availability in culture media, especially nitrogen, stresses the microorganisms and affects cell growth, thus inducing lipid accumulation. This is an interesting step in biodiesel feedstock obtention from microalgae and should be better understood. In this study, four levels of nitrogen concentration in the BG-11 culture medium were evaluated in the growth of the chlorophycean microalga Desmodesmus sp. Both cell growth and lipid content were monitored over 7 days of cultivation, which yielded a final cell density of 33×106 cells mL−1 with an initial NaNO3 concentration of 750 mg L−1 in the medium and a maximum lipid content of 23 % with total nitrogen starvation. It was observed that the microalgae presented high lipid accumulation in the fourth day of cultivation with nitrogen starvation, although with moderate cell growth. Keywords Microalgae . Desmodesmus . Biodiesel . Lipids . Nitrogen starvation
Introduction Biodiesel is a biodegradable fuel, obtained from renewable sources such as animal fats and vegetable oils. Nowadays, the techniques for large-scale biodiesel production from these type of feedstock are highly developed and economically viable [1]. Lately, lipids extracted from microalgae have been considered as an interesting alternative for biodiesel synthesis, since microalgae can present high biomass productivity and its cultivation does not compete with land used in food production [2, 3]. L. F. Rios (*) : B. C. Klein : R. Maciel Filho : M. R. Wolf Maciel Laboratory of Optimization, Design and Advanced Control/Laboratory of Separation Process Development (LOPCA/LDPS), Faculty of Chemical Engineering, State University of Campinas (Unicamp), Av. Albert Einstein, 500, Campinas, SP, Brazil e-mail: [email protected] L. F. Luz Jr. Technology Sector, Chemical Engineering Department, Federal University of Paraná (UFPR), Jardim das Américas, Curitiba, PR, Brazil
Appl Biochem Biotechnol
A great number of microalgae species can be used with this goal. In order to choose the right species for large-scale cultivation, it is important to take into account the trade-off between lipid accumulation and daily biomass productivity. Certain species, such as Botryococcus braunii, may contain up to 75 % lipids in the cell, though presenting relatively slow growth kinetics. Other species, like Chlorella vulgaris and Desmodesmus sp., may attain higher growth rates but with maximum lipid concentrations of 58 and 21 %, respectively [4]. Technologies for microalgae oil production and processing into biodiesel are currently under development, still being considered marginally economically feasible processes for a large-scale biofuel synthesis. Research in these field tends to focus in three main areas: enhancement of microalgae growth, improving extraction step efficiencies [5–7], and novel biodiesel production processes, since the most employed one involves the transesterification of triglycerides with low molecular weight alcohols [8]. In general, biodiesel production cost has two main components: feedstock, which accounts from 60 to 75 % of the total, and process operating costs, accounting for the remainder [9]. Several parameters affect feedstock cost, such as its production and conditioning for the transesterification reaction. In microalgae-based biodiesel, a critical step of the process is lipid obtention, since microalgae must be properly cultivated and harvested from the medium before performing lipid extraction. For the process to become economically attractive, microalgae with high biomass growth associated with increased lipid storage are desirable. This can be achieved through the evaluation of different microalgae species (conventional or genetically modified) and strategies of culture media development. Furthermore, a research on the direct production of fatty acid esters with in situ transesterification aims at the elimination of the lipid extraction step, thus reducing total process cost and chemical consumption [10, 11]. Recent studies show that nitrogen starvation in the culture medium can significantly induce the accumulation of lipids in microalgae [12], despite somewhat hindering cell growth [13]. Several studies have been carried out in order to study this effect in different microalgae species [14–16]. Bearing this in mind, this paper aimed to analyze the growth and the lipid content through fluorescence spectroscopy [17] of the microalga species Desmodesmus sp. cultivated under different levels of nitrogen in BG-11 medium.
Materials and Methods Microalgae Cultures The green microalga species Desmodesmus sp. was provided by the Laboratory of Research on Aquatic Organisms (LAPOA) of the Integrated Group on Aquiculture and Environmental Studies (GIA) of the Federal University of Paraná (UFPR). BG-11 medium was employed in the maintenance of microalgae cultures and in the cultivations of the experimental setup. The chosen medium presents the following composition [18]: NaNO3 (1500 mg L−1), K2HPO4 (40 mg L−1), CaCl2·2H2O (30 mg L−1), Na2CO3 (19 mg L−1), MgSO4·7H2O (8 mg L−1), C6H8O7·H2O (7 mg L−1), ammonium ferric citrate (6 mg L−1), H3BO3 (3 mg L−1), MnCl2·4H2O (2 mg L−1), Na2EDTA·2H2O (0.7 mg L−1), Na2MoO4·2H2O (0.4 mg L−1), ZnSO4·7H2O (0.2 mg L−1), CuSO4·5H2O (0.1 mg L−1), and Co(NO3)2·6H2O (0.05 mg L−1). Before sterilization in an autoclave at 121 °C for 15 min, medium pH was adjusted to 7.5 with a 0.1 N HCl solution.
Appl Biochem Biotechnol
Experimental Setup In order to assess the influence of nitrogen concentration on lipid accumulation, an experimental layout of three modifications of the BG-11 medium was performed, as shown in Table 1. Inocula for these cultivations were produced in 1-L Erlenmeyer flasks, with a working volume of 600 mL of BG-11 medium, under light flux of 62 μE m−2 s−1, photoperiod of 12 h (light-dark), mean temperature of 27 °C, and forced aeration with atmospheric air. Inocula cultivations were maintained until the late exponential growth phase (day 6), and the biomass was harvested by centrifugation at 1843×g for 15 min (Eppendorf, model 5810R). The recovered biomass was washed with distilled water several times for salt removal and divided into four equal parts. Each fraction was inoculated in an Erlenmeyer flask containing one of the modified BG-11 media (0, 25, 50, and 100 N). Analytical Methods Biomass growth was monitored periodically through cell counting with a Neubauer chamber in a microscope (Olympus, model CX21). All results are given in millions of cells per milliliter. Images of the cultivations were made with a microscope (Leica, model DMLMem) at ×500 transmitted amplification. Microalgae lipid contents were qualitatively analyzed through fluorescence according to the modified method of Chen et al. [19]. Measurements were carried out in a fluorometer (HITACHI, model F4500) with an excitation wavelength of 515 nm in an emission range of 515–800 nm. Daily samples were taken from the cultivations and incubated for 12 h with analytical-grade dimethyl sulfoxide (DMSO). After incubation, microalgae cells were determined through direct counting, maintaining in every sample a concentration of 2 million cells mL−1 for greater consistency and better results comparison. Afterwards, 3 μL of Nile red (9-diethylamino-5-benzo[α]phenoxazinone) diluted in acetone (0.1 mg mL−1) was added to a 2-mL aliquot of each sample and finally incubated for 10 min at 37 °C before each measurement. Lipid extraction was carried out through the Bligh and Dyer method [20] with the biomass produced within 7 days of cultivation. Initially, 50-mg samples of dry microalgae biomass were placed into an ultrasound water bath (Unique, model USC-2800) at a frequency of 40 kHz for 15 min in order to disrupt cell walls. The samples were treated with water and a 2:1 methanol/chloroform mixture and agitated in an orbital mixer for 25 min. The suspension was then centrifuged (Eppendorf, model 5810R) at 4500×g for 10 min, yielding three distinct phases: top, with water and methanol; middle, a semisolid protein disk; and bottom, an organic phase with lipids dissolved in chloroform. Afterwards, chloroform was removed from the organic phase through nitrogen evaporation. To ensure chloroform elimination, the lipid samples were placed in an oven at 105 °C for 24 h. Lipid mass fractions were determined by gravimetry upon reaching constant weight in a vacuum desiccator. Table 1 Variations of the culture medium employed in the experimental layout Designation (N)
Initial NaNO3 concentration (mg L−1)
0
0
25
375
50
750
100 (no modification)
1500
Appl Biochem Biotechnol
Fig. 1 Photomicrography of the Desmodesmus sp. strain employed in the study
Results and Discussion Growth Analysis Figure 1 shows a microphotography of the Desmodesmus sp. strain used in the study. A mean diameter of 6.25 μm was calculated from measurements of 30 different cells. Figure 2a shows the growth curve for the microalgae species in BG-11 medium, without alteration in the initial nitrogen content (100 N). All kinetics exhibit the characteristic S-curve shape for microorganism growth: from the start until day 2, a period of adaptation to the culture medium (lag phase) is observed, and from day 2 until day 10, an exponential growth
(a)
(b) 55 50
Million of cells/mL
45 40 35 30 25 20 15 10 5 0 0
2
4
6
8
10
12
Time (days)
Fig. 2 Desmodesmus sp. growth curve a in unmodified BG-11 medium for 12 days (inoculum) and b in BG-11 media with different initial NaNO3 concentrations (0, 25, 50, and 100 N) for 7 days
Appl Biochem Biotechnol
(a)
(b)
Fig. 3 Growth kinetics of Desmodesmus sp. growth a in 50 and 100 N BG-11 media and b in mixed-media growth (50 and 100 to 50 N)
phase (log phase) appears. Inocula biomass harvest was carried out in the sixth day of cultivation in order to avoid the occurrence of a lag phase in the cultivations of the experimental layout. In Fig. 2b, the growth curves for the four devised cultivations with different initial NaNO3 concentrations are shown. Initially, all cultivations presented similar growth behavior. Although with a slight increase in cell number in the 0 N (0 % of nitrogen) batch until day 4, the final biomass density was the lowest among all experiments. The nitrogen deprivation study showed that it was possible to obtain a final microalgae concentration 16×107 cells mL−1 higher in a 50 N medium in comparison to the unmodified BG-11 medium. This fact is also interesting as it hints to the possibility of reducing the amount of NaNO3 in the BG-11 culture medium without impacting (and even favoring) microalgae development, thus saving nutrient consumption. Two supplementary cultivations were carried out in 50 and 100 N BG-11 media and monitored. Growth kinetics is shown in Fig. 3a. It can be observed that both batches presented similar growth curves, with the 100 N medium reaching higher final cell density. In addition, in the 6th day of the 100 N cultivation, half of the recovered biomass was inoculated in a 50 N medium. Growth kinetics for this arrangement (100 N/50 N) and for an unmodified 100 N BG11 medium are shown in Fig. 3b. The batch with limited nitrogen attained a slightly higher final cell density. Lipid Quantification At the term of 12 days, lipids were extracted from the biomass recovered from each cultivation of the experimental layout through the application of the Bligh and Dyer method. Results are shown in Table 2. It can be seen that lipid content in the 0 N cultivation is the highest among Table 2 Lipid content of the four cultivations of the experimental layout Designation (N)
Lipid content (%), dry basis
0
23.0
25
5.8
50
11.0
100 (no modification)
15.0
Appl Biochem Biotechnol
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(b) 120
100
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80
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20
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750
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Fig. 4 Fluorescence spectra of nitrogen-limited cultivations: a day 0, b day 1, c day 2, d day 3, e day 4, f day 5, g day 6, and h day 7
Appl Biochem Biotechnol
the batches, with a percentage in accord with the maximum values found in the scientific literature [4]. Other cultivations presented mixed results, with values ranging from 5.8 % (25 N) to 15 % (100 N) of lipid content. Fluorescence spectroscopy spectra of the four cultivations with different levels of nitrogen are shown in Fig. 4. Lipids were analyzed in the wavelengths from 540 to 640 nm and chlorophyll in the range of 640 to 700 nm. Figure 4a indicates the start of microalgae growth (day 0), where only chlorophyll can be identified. Lipids began to appear in fluorescence spectra from day 1 of the cultivations. In Fig. 4b, it can be seen that the batch with 0 N BG-11 medium presented the highest lipid content among the experiments. At days 2 and 3 (Fig. 4c, d), spectra showed different patterns, since nitrogen could be detected in all of the batches. The experiment with 25 N BG-11 medium presented the highest lipid amount. In Fig. 4e, day 4 of the experiments, the lipid content in the 0 N BG-11 batch again was the highest, with the 25 N BG-11 having the greatest chlorophyll content. Only in the fifth day of cultivation (Fig. 4f), the 100 N BG-11 batch presented an increase in the lipid fraction, which was maintained up to day 6. At day 7 of the cultivation (Fig. 4h), no apparent changes in lipid content were observed, although a slight increase in chlorophyll amount has been seen. In essence, the analyses carried out with the fluorescence spectroscopy showed that lipids varied considerably during each day of cultivation and this fluctuation is influenced by NaNO3 concentration in the culture medium.
Conclusions Nitrogen limitation on Desmodesmus sp. cultivations showed to be efficient in influencing both cell growth and lipid accumulation. The highest growth was observed for the 50 N BG-11 medium, with a final cell count of 33×106 cells mL−1. In terms of lipid content, a maximum amount of 23 % (dry basis) was obtained with total nitrogen deprivation (0 N BG-11 medium), although with considerable growth hindrance. From this study, a strategy for maximization of lipid productivity with Desmodesmus sp. can be designed: (1) the inoculum should be cultivated in 100 N BG-11 medium, with its biomass being harvested at day 6 of the batch, and (2) the biomass should, then, be placed in a fully nitrogen-deprived culture medium (0 N BG-11) and cultivated for 4 days.
References 1. Santori, G., Di Nicola, G., Moglie, M., & Polonara, F. (2012). Applied Energy, 92, 109–132. 2. Hossain, S. A., Salleh, A., Boyce, A. N., Chowdhury, P., & Naquiuddin, M. (2008). American Journal of Biochemistry and Biotechnology, 4, 250–254. 3. Pragya, N., Pandey, K. K., & Sahoo, P. (2013). Renewable and Sustainable Energy Reviews, 24, 159–171. 4. Mata, T., Martins, A., & Caetano, N. (2010). Renewable and Sustainable Energy Reviews, 14, 217–232. 5. Lee, J.-Y., Yoo, C., Jun, S.-Y., Ahnn, C.-Y., & Oh, H.-M. (2010). Bioresource Technology, 101, 575–577. 6. Halim, R., Gladman, B., Danquah, M. K., & Webley, P. A. (2011). Bioresource Technology, 102, 178–185. 7. Halim, R., Danquah, M. K., & Webley, P. A. (2012). Biotechnology Advances, 30, 709–732. 8. Hidalgo, P., Toro, C., Ciudad, G., & Navia, R. (2013). Reviews in Environmental Science and Biotechnology, 12, 179–199. 9. Huang, G., Chen, F., Wei, D., Zhang, X., Chen, G. (2010). Applied Energy, 38–46. 10. Ehimen, E. A., Sun, Z. F., & Carrington, C. G. (2010). Fuel, 89, 677–684. 11. Haas, M. J., & Wagner, K. (2011). Journal of Lipid Science and Technology, 113, 1219–1229.
Appl Biochem Biotechnol 12. Ruiz-Marin, A., Mendoza-Espinosa, L. G., & Stephenson, T. (2010). Bioresource Technology, 101, 58–64. 13. Li, Y., Horsman, M., Wang, B., Wu, N. (2008). Biotechnological Products and Process Engineering, 629– 636. 14. Adams, C., Godfrey, V., Wahlen, B., Seefeldt, L., & Bugbee, B. (2013). Bioresource Technology, 131, 188– 194. 15. Dragone, G., Fernandes, A. P., Abreu, A. A., & Teixeira, J. A. (2011). Applied Energy, 88, 3331–3335. 16. Klok, A. J., Martens, D. E., Wijffels, R. H., & Lamers, P. P. (2013). Bioresource Technology, 134, 233–243. 17. Elsey, D., Jameson, D., Raleigh, B., & Cooney, M. (2007). Journal of Microbiological Methods, 68, 639– 642. 18. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., & Stanier, R. Y. (1979). Journal of General Microbiology, 111, 1–61. 19. Chen, W., Zhang, C., Song, L., Sommerfeld, M., & Hu, Q. (2009). Journal of Microbiological Methods, 77, 41–47. 20. Bligh, E. G., & Dyer, W. J. (1959). Canadian Journal of Biochemistry and Physiology, 8, 911–917.
Nitrogen Starvation for Lipid Accumulation in the Microalga Species Desmodesmus sp. L. F. Rios & B. C. Klein & L. F. Luz Jr. & R. Maciel Filho & M. R. Wolf Maciel
Received: 6 May 2014 / Accepted: 2 October 2014 # Springer Science+Business Media New York 2014
Abstract Recently, to obtain lipids from microalgae has been the object of extensive research, since it is viewed as a promising feedstock for biodiesel production, especially when compared with crops such as soybean and sunflower, in terms of theoretical performance. The reduction of nutrient availability in culture media, especially nitrogen, stresses the microorganisms and affects cell growth, thus inducing lipid accumulation. This is an interesting step in biodiesel feedstock obtention from microalgae and should be better understood. In this study, four levels of nitrogen concentration in the BG-11 culture medium were evaluated in the growth of the chlorophycean microalga Desmodesmus sp. Both cell growth and lipid content were monitored over 7 days of cultivation, which yielded a final cell density of 33×106 cells mL−1 with an initial NaNO3 concentration of 750 mg L−1 in the medium and a maximum lipid content of 23 % with total nitrogen starvation. It was observed that the microalgae presented high lipid accumulation in the fourth day of cultivation with nitrogen starvation, although with moderate cell growth. Keywords Microalgae . Desmodesmus . Biodiesel . Lipids . Nitrogen starvation
Introduction Biodiesel is a biodegradable fuel, obtained from renewable sources such as animal fats and vegetable oils. Nowadays, the techniques for large-scale biodiesel production from these type of feedstock are highly developed and economically viable [1]. Lately, lipids extracted from microalgae have been considered as an interesting alternative for biodiesel synthesis, since microalgae can present high biomass productivity and its cultivation does not compete with land used in food production [2, 3]. L. F. Rios (*) : B. C. Klein : R. Maciel Filho : M. R. Wolf Maciel Laboratory of Optimization, Design and Advanced Control/Laboratory of Separation Process Development (LOPCA/LDPS), Faculty of Chemical Engineering, State University of Campinas (Unicamp), Av. Albert Einstein, 500, Campinas, SP, Brazil e-mail: [email protected] L. F. Luz Jr. Technology Sector, Chemical Engineering Department, Federal University of Paraná (UFPR), Jardim das Américas, Curitiba, PR, Brazil
Appl Biochem Biotechnol
A great number of microalgae species can be used with this goal. In order to choose the right species for large-scale cultivation, it is important to take into account the trade-off between lipid accumulation and daily biomass productivity. Certain species, such as Botryococcus braunii, may contain up to 75 % lipids in the cell, though presenting relatively slow growth kinetics. Other species, like Chlorella vulgaris and Desmodesmus sp., may attain higher growth rates but with maximum lipid concentrations of 58 and 21 %, respectively [4]. Technologies for microalgae oil production and processing into biodiesel are currently under development, still being considered marginally economically feasible processes for a large-scale biofuel synthesis. Research in these field tends to focus in three main areas: enhancement of microalgae growth, improving extraction step efficiencies [5–7], and novel biodiesel production processes, since the most employed one involves the transesterification of triglycerides with low molecular weight alcohols [8]. In general, biodiesel production cost has two main components: feedstock, which accounts from 60 to 75 % of the total, and process operating costs, accounting for the remainder [9]. Several parameters affect feedstock cost, such as its production and conditioning for the transesterification reaction. In microalgae-based biodiesel, a critical step of the process is lipid obtention, since microalgae must be properly cultivated and harvested from the medium before performing lipid extraction. For the process to become economically attractive, microalgae with high biomass growth associated with increased lipid storage are desirable. This can be achieved through the evaluation of different microalgae species (conventional or genetically modified) and strategies of culture media development. Furthermore, a research on the direct production of fatty acid esters with in situ transesterification aims at the elimination of the lipid extraction step, thus reducing total process cost and chemical consumption [10, 11]. Recent studies show that nitrogen starvation in the culture medium can significantly induce the accumulation of lipids in microalgae [12], despite somewhat hindering cell growth [13]. Several studies have been carried out in order to study this effect in different microalgae species [14–16]. Bearing this in mind, this paper aimed to analyze the growth and the lipid content through fluorescence spectroscopy [17] of the microalga species Desmodesmus sp. cultivated under different levels of nitrogen in BG-11 medium.
Materials and Methods Microalgae Cultures The green microalga species Desmodesmus sp. was provided by the Laboratory of Research on Aquatic Organisms (LAPOA) of the Integrated Group on Aquiculture and Environmental Studies (GIA) of the Federal University of Paraná (UFPR). BG-11 medium was employed in the maintenance of microalgae cultures and in the cultivations of the experimental setup. The chosen medium presents the following composition [18]: NaNO3 (1500 mg L−1), K2HPO4 (40 mg L−1), CaCl2·2H2O (30 mg L−1), Na2CO3 (19 mg L−1), MgSO4·7H2O (8 mg L−1), C6H8O7·H2O (7 mg L−1), ammonium ferric citrate (6 mg L−1), H3BO3 (3 mg L−1), MnCl2·4H2O (2 mg L−1), Na2EDTA·2H2O (0.7 mg L−1), Na2MoO4·2H2O (0.4 mg L−1), ZnSO4·7H2O (0.2 mg L−1), CuSO4·5H2O (0.1 mg L−1), and Co(NO3)2·6H2O (0.05 mg L−1). Before sterilization in an autoclave at 121 °C for 15 min, medium pH was adjusted to 7.5 with a 0.1 N HCl solution.
Appl Biochem Biotechnol
Experimental Setup In order to assess the influence of nitrogen concentration on lipid accumulation, an experimental layout of three modifications of the BG-11 medium was performed, as shown in Table 1. Inocula for these cultivations were produced in 1-L Erlenmeyer flasks, with a working volume of 600 mL of BG-11 medium, under light flux of 62 μE m−2 s−1, photoperiod of 12 h (light-dark), mean temperature of 27 °C, and forced aeration with atmospheric air. Inocula cultivations were maintained until the late exponential growth phase (day 6), and the biomass was harvested by centrifugation at 1843×g for 15 min (Eppendorf, model 5810R). The recovered biomass was washed with distilled water several times for salt removal and divided into four equal parts. Each fraction was inoculated in an Erlenmeyer flask containing one of the modified BG-11 media (0, 25, 50, and 100 N). Analytical Methods Biomass growth was monitored periodically through cell counting with a Neubauer chamber in a microscope (Olympus, model CX21). All results are given in millions of cells per milliliter. Images of the cultivations were made with a microscope (Leica, model DMLMem) at ×500 transmitted amplification. Microalgae lipid contents were qualitatively analyzed through fluorescence according to the modified method of Chen et al. [19]. Measurements were carried out in a fluorometer (HITACHI, model F4500) with an excitation wavelength of 515 nm in an emission range of 515–800 nm. Daily samples were taken from the cultivations and incubated for 12 h with analytical-grade dimethyl sulfoxide (DMSO). After incubation, microalgae cells were determined through direct counting, maintaining in every sample a concentration of 2 million cells mL−1 for greater consistency and better results comparison. Afterwards, 3 μL of Nile red (9-diethylamino-5-benzo[α]phenoxazinone) diluted in acetone (0.1 mg mL−1) was added to a 2-mL aliquot of each sample and finally incubated for 10 min at 37 °C before each measurement. Lipid extraction was carried out through the Bligh and Dyer method [20] with the biomass produced within 7 days of cultivation. Initially, 50-mg samples of dry microalgae biomass were placed into an ultrasound water bath (Unique, model USC-2800) at a frequency of 40 kHz for 15 min in order to disrupt cell walls. The samples were treated with water and a 2:1 methanol/chloroform mixture and agitated in an orbital mixer for 25 min. The suspension was then centrifuged (Eppendorf, model 5810R) at 4500×g for 10 min, yielding three distinct phases: top, with water and methanol; middle, a semisolid protein disk; and bottom, an organic phase with lipids dissolved in chloroform. Afterwards, chloroform was removed from the organic phase through nitrogen evaporation. To ensure chloroform elimination, the lipid samples were placed in an oven at 105 °C for 24 h. Lipid mass fractions were determined by gravimetry upon reaching constant weight in a vacuum desiccator. Table 1 Variations of the culture medium employed in the experimental layout Designation (N)
Initial NaNO3 concentration (mg L−1)
0
0
25
375
50
750
100 (no modification)
1500
Appl Biochem Biotechnol
Fig. 1 Photomicrography of the Desmodesmus sp. strain employed in the study
Results and Discussion Growth Analysis Figure 1 shows a microphotography of the Desmodesmus sp. strain used in the study. A mean diameter of 6.25 μm was calculated from measurements of 30 different cells. Figure 2a shows the growth curve for the microalgae species in BG-11 medium, without alteration in the initial nitrogen content (100 N). All kinetics exhibit the characteristic S-curve shape for microorganism growth: from the start until day 2, a period of adaptation to the culture medium (lag phase) is observed, and from day 2 until day 10, an exponential growth
(a)
(b) 55 50
Million of cells/mL
45 40 35 30 25 20 15 10 5 0 0
2
4
6
8
10
12
Time (days)
Fig. 2 Desmodesmus sp. growth curve a in unmodified BG-11 medium for 12 days (inoculum) and b in BG-11 media with different initial NaNO3 concentrations (0, 25, 50, and 100 N) for 7 days
Appl Biochem Biotechnol
(a)
(b)
Fig. 3 Growth kinetics of Desmodesmus sp. growth a in 50 and 100 N BG-11 media and b in mixed-media growth (50 and 100 to 50 N)
phase (log phase) appears. Inocula biomass harvest was carried out in the sixth day of cultivation in order to avoid the occurrence of a lag phase in the cultivations of the experimental layout. In Fig. 2b, the growth curves for the four devised cultivations with different initial NaNO3 concentrations are shown. Initially, all cultivations presented similar growth behavior. Although with a slight increase in cell number in the 0 N (0 % of nitrogen) batch until day 4, the final biomass density was the lowest among all experiments. The nitrogen deprivation study showed that it was possible to obtain a final microalgae concentration 16×107 cells mL−1 higher in a 50 N medium in comparison to the unmodified BG-11 medium. This fact is also interesting as it hints to the possibility of reducing the amount of NaNO3 in the BG-11 culture medium without impacting (and even favoring) microalgae development, thus saving nutrient consumption. Two supplementary cultivations were carried out in 50 and 100 N BG-11 media and monitored. Growth kinetics is shown in Fig. 3a. It can be observed that both batches presented similar growth curves, with the 100 N medium reaching higher final cell density. In addition, in the 6th day of the 100 N cultivation, half of the recovered biomass was inoculated in a 50 N medium. Growth kinetics for this arrangement (100 N/50 N) and for an unmodified 100 N BG11 medium are shown in Fig. 3b. The batch with limited nitrogen attained a slightly higher final cell density. Lipid Quantification At the term of 12 days, lipids were extracted from the biomass recovered from each cultivation of the experimental layout through the application of the Bligh and Dyer method. Results are shown in Table 2. It can be seen that lipid content in the 0 N cultivation is the highest among Table 2 Lipid content of the four cultivations of the experimental layout Designation (N)
Lipid content (%), dry basis
0
23.0
25
5.8
50
11.0
100 (no modification)
15.0
Appl Biochem Biotechnol
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Fig. 4 Fluorescence spectra of nitrogen-limited cultivations: a day 0, b day 1, c day 2, d day 3, e day 4, f day 5, g day 6, and h day 7
Appl Biochem Biotechnol
the batches, with a percentage in accord with the maximum values found in the scientific literature [4]. Other cultivations presented mixed results, with values ranging from 5.8 % (25 N) to 15 % (100 N) of lipid content. Fluorescence spectroscopy spectra of the four cultivations with different levels of nitrogen are shown in Fig. 4. Lipids were analyzed in the wavelengths from 540 to 640 nm and chlorophyll in the range of 640 to 700 nm. Figure 4a indicates the start of microalgae growth (day 0), where only chlorophyll can be identified. Lipids began to appear in fluorescence spectra from day 1 of the cultivations. In Fig. 4b, it can be seen that the batch with 0 N BG-11 medium presented the highest lipid content among the experiments. At days 2 and 3 (Fig. 4c, d), spectra showed different patterns, since nitrogen could be detected in all of the batches. The experiment with 25 N BG-11 medium presented the highest lipid amount. In Fig. 4e, day 4 of the experiments, the lipid content in the 0 N BG-11 batch again was the highest, with the 25 N BG-11 having the greatest chlorophyll content. Only in the fifth day of cultivation (Fig. 4f), the 100 N BG-11 batch presented an increase in the lipid fraction, which was maintained up to day 6. At day 7 of the cultivation (Fig. 4h), no apparent changes in lipid content were observed, although a slight increase in chlorophyll amount has been seen. In essence, the analyses carried out with the fluorescence spectroscopy showed that lipids varied considerably during each day of cultivation and this fluctuation is influenced by NaNO3 concentration in the culture medium.
Conclusions Nitrogen limitation on Desmodesmus sp. cultivations showed to be efficient in influencing both cell growth and lipid accumulation. The highest growth was observed for the 50 N BG-11 medium, with a final cell count of 33×106 cells mL−1. In terms of lipid content, a maximum amount of 23 % (dry basis) was obtained with total nitrogen deprivation (0 N BG-11 medium), although with considerable growth hindrance. From this study, a strategy for maximization of lipid productivity with Desmodesmus sp. can be designed: (1) the inoculum should be cultivated in 100 N BG-11 medium, with its biomass being harvested at day 6 of the batch, and (2) the biomass should, then, be placed in a fully nitrogen-deprived culture medium (0 N BG-11) and cultivated for 4 days.
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