A MULTISTAGE GRADUAL NITROGENREDUCTION STRATEGY FOR INCREASED LIPID PRODUCTIVITY AND NITROGEN REMOVAL IN WASTEWATER USING Chlorella vulgaris AND Scenedesmus obliquus

Abstract

Chlorella vulgaris and Scenedesmus obliquuswere grown in artificial-wastewater using a new nitrogen-limitation strategy aimed at increasing lipid productivity. This strategy consisted in a multi-stage process with sequential reduction of N-NH₄ concentration (from 90 to 60, 40, and 20 mg.L^-1) to promote a balance between cell growth and lipid accumulation. Lipid productivity was compared against a reference process consisting of nitrogen reduction in two stages, where the nitrogen concentration was suddenly reduced from 90 mg.L^-1 to three different concentrations (10, 20, and 30 mg.L^-1). In the multi-stage mode, only C. vulgaris exhibited a net lipid-productivity increase. Lipid content of S. obliquus did not present a significant increase, thus decreasing lipid productivity. The highest lipid productivities were observed in the two-stage mode for both S. obliquus and C. vulgaris (194.9 and 133.5 mg.L^-1.d^-1, respectively), and these values are among the highest reported in the literature to date.

Keywords
Nitrogen limitation; Lipid productivity; Multistage; Microalgae; Biodiesel

INTRODUCTION

Biodiesel is one of the liquid biofuels that has received most interest due to its many advantages over fossil diesel. Currently, it is commercially produced from oleaginous plants, such as soy, African palm, and rapeseed. However, two major problems for the industrial production of biodiesel are feedstock availability, and land competition (direct or indirect) with food crops. As a response to these challenges, microalgae have been explored as a promising oil supply: they have higher photosynthetic efficiencies than terrestrial crops, exhibit higher biomass production rates per hectare, and can be used as a tertiary wastewater treatment. Many studies have shown that some microalgae species can store large amounts of triacylglycerides (TAGs) consisting of a mixture of saturated and unsaturated fatty-acid chains (C₁₂ to C₂₂), which makes them appropriate for biodiesel production (Borowitzka, 1991).

Some microalgae species have high lipid content (20-50% of dry cell weight) and it is possible to increase it by controlling various biotic and abiotic factors of the culture, such as light intensity and intermittence, temperature, salinity, CO₂addition, agitation regime, and harvesting methods (Hu et al., 2008; Chiu et al., 2009; Widjaja et al. 2009). One of the most studied factors for increasing the lipid content is nutrient limitation; in particular, nitrogen and phosphorous limitation have been reported to increase lipid content and to shift the lipid composition towards TAGs (Widjaja et al., 2009), although little is known about the underlying mechanisms (Li et al.,2012). The microalgae Chlorella and Scenedesmus are often used in biodiesel-production studies, and they present the ability to grow in wastewater. Illman et al. (2000) reported that Chlorella vulgaris accumulated 14-30% w/w of lipids under N-sufficient conditions. The lipid content of C. vulgaris increased from 20% to 43% of dry cell weight under nitrogen starvation conditions (Mujtaba et al., 2012). Scott et al. (2010) reported lipid contents of up to 70% for the same species, when cultivated under nitrogen deficiency. Furthermore, response to nutrient limitation has been shown to be species-specific (Griffiths et al.,2011).

When grown under nutrient limitation, lipid content is expected to increase at the expense of cellular growth. This occurs because, under limited N concentration, protein synthesis required for cell growth is inhibited, leaving an excess of carbon from photosynthesis that is redirected to the metabolic paths of lipid storage and starch production (Li et al.,2012; Scott et al.,2010). Lipid productivity P_L (grams of lipids per liter of culture per day) is the most important variable to maximize the biodiesel production from microalgae cultures (Khozin and Cohen, 2006; Rodolfi et al., 2009), since it represents the coupled effect of increased lipid content w (grams of lipids per grams of dry biomass) and reduced biomass concentration X (grams of dry biomass per liter of culture) in N-limited cultures.

Because of their high lipid productivity and adequate fatty-acid profile, C. vulgaris and S. obliquus have been identified as two species with great potential for biodiesel production (Griffiths et al., 2011). Several works have reported lipid productivities of these two species as high as 180 mg.L^-1.d^-1 and 140 mg.L^-1.d^-1 for C. vulgaris and S. obliquus, respectively (Table 1). These productivity values were achieved under nitrogen-limited culture conditions (Widjaja et al., 2009; Mujtaba et al., 2012; Griffiths et al., 2011; Gouveia and Oliveira, 2009; Feng et al., 2011; Pranveenkumar et al., 2012; Lv et al., 2010; Ho et al., 2010; Converti et al., 2009; Ho et al., 2012).

Table 1
Reported lipid productivities for C. vulgaris and S. obliquus in photoautotrophic cultures.

Species	PL (mg L-1d-1)	Culture conditions and reference
C. vulgaris	180.0	Nitrogen limitation (Gouveia and Oliveira, 2009)
	147.0	Semi-continuous cultivation in bubble column photobioreactors (Feng et al., 2011)
	77.1	Nitrogen limitation (Mujtaba et al., 2012)
	67.0	Nitrogen limitation (Griffiths et al., 2011)
	54.0	Nitrogen starvation of Chlorella sp. BUM11008 (Praveenkumar et al.,2012).
	40.0	Optimized productivity limiting nitrogen source, adding CO2 and varying the light intensity (Lv et al.,2010)
	16.9	Nitrogen starvation (Widjaja et al., 2009)
	20.4	Reduced nitrate culture (Converti et al.,2009)
S. obliquus	140.4	Five-day nitrogen starvation in S. obliquus CNW-N (Ho et al., 2012)
	106.0	Nitrogen limitation (Griffiths et al., 2011)
	90.0	Nitrogen limitation (Gouveia and Oliveira, 2009)
	78.73	Nine-day nitrogen starvation in S. obliquus CNW-N (Ho et al., 2010)

Using a two-stage cultivation mode, Rodolfi et al. (2009) grew Nannochloropsissp. F&M-M24 in nitrogen-rich medium, and then diluted with fresh, low-nitrogen medium, increasing lipid productivity to 200 mg.L^-1.d^-1. On the other hand, Stephenson et al. (2012) reported that the most effective strategy to increase the productivity of TAGs in C. vulgaris was by continuing the cultivation until nitrogen depletion (in a single stage), rather than transferring the culture to fresh medium without nitrogen. Also, Mujtaba et al. (2012) reported a constant lipid content (14-16% w/w) and lipid productivity in C. vulgaris when growing the microalgae for 5 and 7 days using two cultivation modes: a single stage, and continuous addition of nitrogen in batch reactors. These results show that reducing the N concentration in the medium will result in an increase of lipid content in the microalgae, but the net lipid productivity may be the same, or even lower than in single batch culture, as a result of the reduction in the growth rate.

Furthermore, Feng et al.(2011) reported high lipid productivities in a semi-continuous culture of C. vulgaris in aerated-column reactors.

They showed that 50% v/v dilutions with fresh medium every 24 h for three days, resulted in a lipid productivity of 147 mg.L^-1.d^-1, compared to 79 mg.L^-1.d^-1 obtained with daily replacement of 75% v/v with fresh medium. The lower lipid productivity in the second case was related to the reduced growth rates, since lipid content remained almost constant in both processes (38-42% w/w, respectively).

Based on all this evidence, a cultivation strategy consisting of sequential stages of gradual reduction of N concentration in the medium is analyzed in this study. In the design of microalgal-oil production processes it is apparent that lipid productivity is more important than lipid content only. Therefore, a strategy to increase the lipid productivity in autotrophic cultures may require balancing lipid content and growth rate through a multistage process, involving the gradual reduction of N concentration in the culture medium. Thus, two autotrophic cultivation modes of C. vulgaris and S. obliquus were compared: a multistage process (or gradual nitrogen reduction), and the most studied two-stage process (or sudden nitrogen reduction).

MATERIALS AND METHODS

Strain Selection and Culture Media

Strains of Chlorella vulgaris and Scenedesmus obliquus were obtained from the culture collection of the Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Mexico. These species were selected due to their proven capacity to grow in wastewater, and having high N and P removal efficiencies (Ruiz-Marin et al. 2010). For the acclimation, both species were grown in culture media with a composition similar to that in the effluent of the primary treatment of an urban wastewater-treatment plant (Ruiz-Marin et al. 2010), as follows: 7 mg NaCl, 4 mg CaCl₂, 2 mg MgSO₄·7H₂O, 15 mg KH₂PO₄, 115.6 mg NH₄Cl, all dissolved in 1 L of distilled water. Trace metals and vitamins were added according to the guidelines for medium f/2 (Guillar and Ryther 1962). During acclimation (1 month), both microalgae were transferred to fresh culture media every seven days at 28±1 °C and light intensity of 100 µE.m^-2.s^-1.

Two-Stage (TS) Nitrogen Reduction Cultures

The cultures were operated in 3 L cylindrical bioreactors made of transparent polyethylene terephthalate (PETE) containing 2 L of operational volume of culture medium. The reactors were pre-washed with a chlorine solution to prevent bacterial contamination. The treatments were run with an initial cell density of 2×10⁶ cells.mL^-1 and aerated at 0.4 L.L^-1.min^-1, under a continuous illumination regime at 120 µE.m^-2.s^-1 (cool-white fluorescent lamps) and at a constant temperature of 28 ± 1 °C.

In most studies using a two-stage method for nutrient limitation, the first stage is a nutrient-rich culture with the purpose of achieving a high biomass concentration, while depleting the nitrogen in the medium; for this reason, the biomass is harvested at the end of the logarithmic phase of growth (centrifugation and precipitation being the most commonly used methods). The harvested biomass is then resuspended in fresh medium containing a low concentration or absence of nitrogen (Widjaja et al., 2009; Griffiths et al., 2011; Converti et al., 2009; Li et al., 2008). For practical purposes, especially in larger capacity reactors, it is more convenient to replace the depleted medium from the first stage with fresh medium containing a lower initial nitrogen-concentration (Converti et al., 2009; Li et al., 2008; Zhila et al., 2005; Tan and Lin, 2011). In this study, the latter approach was taken: the microalgae were first grown in a nitrogen-rich medium (90 mg.L^-1 in the first stage), using an operational volume of 1 L; at the end of the logarithmic phase of growth, 1 L of fresh medium was added (in order to achieve a 50% v/v dilution of the biomass). The residual nitrogen from the first stage was taken into account for adjusting the nitrogen concentration in the fresh medium in order to achieve initial concentrations in the second stage of 30, 20, or 10 mg.L^-1 of N-NH₄. At the end of each stage, measurements were made to determine nitrogen and biomass concentration and lipid content, as described in the following sections.

Multi-stage (MS) Nitrogen Reduction Cultures

The experimental system was the same as in the TS cultures. Initially, stock suspensions of C. vulgaris and S. obliquus of 2×10⁶ cells.mL^-1 were grown under nitrogen-rich conditions (90 mg.L^-1 N-NH₄). After six days, the second stage was initiated by replacing 50% v/v of the depleted medium, and the N-concentration in the fresh medium was adjusted to achieve 60 mg.L^-1of N-NH₄ in the resulting medium. This procedure was repeated twice more at the end of each exponential phase of growth, starting a third and a fourth stage with initial N-NH₄ concentrations of 40 and 20 mg.L^-1, respectively.

Determination of N-NH⁴ Content, Cell Density, and Cell Dry-Weight

Every 12 h, 50 mL of water sample was collected for analysis. The concentration of NH₄ was determined according to standard methods (APHA, 1995). The number of algal cells was determined using a Neubauer chamber Hemacytometer 0.1 mm in depth. For the determination of ash-free dry weight, 20 mL of culture was filtered through a GF-C glass fiber filter previously rinsed with distilled water, and incinerated at 470 °C for 4 h. The samples were dried at 120 °C until constant weight for 2 h in a conventional oven, and then in a muffle furnace at 450 °C for 3 h.

Lipid Extraction and Fatty Acid Composition Analysis

At the end of each treatment, approximately 1 L of culture was collected and then centrifuged at 4.500 rpm and 14 °C for 15 min. The recovered biomass was frozen at -4.0 °C for 48 h, and then lyophilized for 3-5 days; the dry biomass was stored under refrigeration at 0 °C. Total lipids were extracted following the dry extraction procedure described by Feng et al. (2011), and then quantified using the method by Pande et al.(1963) using a tripalmitin standard (99%, Sigma-Aldrich).

For the analysis of the fatty acids in the microalgal oil, approximately 10 mg of biomass was mixed with 4 mL of methanol, 2 mL of chloroform, and 0.5 mL of distilled water; the mixture was first sonicated for 15-30 min at 10 °C, and then centrifuged at 4000 rpm for 15 min. The lipid-solvent phase was recovered by washing the upper layer with 2 mL of distilled water, centrifuging and drying with nitrogen-gas. The microalgal lipids were transesterified with 2.5 mL of a HCl:CH₃OH solution (5% v/v) for 2.5 h at 85 °C (Sato and Murata, 1988). The obtained fatty-acid methyl esters (FAMEs) were extracted with 3 mL hexane, washed with distilled water and dried with nitrogen gas, obtaining samples of about 0.5 mL, which were re-suspended in 1 mL of hexane.

FAME profiles were obtained using an Agilent Technology 7890 gas chromatograph (GC). One microliter of the FAME-hexane solution was injected into the GC equipped with a flame ionization detector (FID) and the separation was performed on a DB23 column (60 m length, 0.32 mm ID, 0.25 µm thickness) with helium as the carrier gas. The temperature of the injector and detector was 250 °C. The temperature program started at 120 °C, holding for 5 min, then increasing at 10 °C.min^-1 until 180 °C, where it was held for 30 min, and then increased to 210 °C at 10 °C.min^-1 (held 21 min). A calibration curve was prepared for all FAMEs by injecting known concentrations of an external standard mixture comprising 37 FAMEs (Supelco, Bellefonte, PA, USA), obtaining a correlation coefficient equal to or greater than 95% in all cases.

Calculation of the Specific Growth Rate and Lipid Productivity

The rate of biomass production (or simply the growth rate) can be expressed in terms of the specific growth rate µ so that dX / dt = µX . Since it remains constant during the logarithmic phase:

where t₁ and t₂ are cultivation times during the logarithmic phase. The lipid productivity is given by (P_L = d(wX)/ dt ; in batch cultures the overall lipid productivity can be approximated by:

where Δ(wX) represents the accumulated lipids from inoculation to harvest, which occurs in the time Δt.

RESULTS AND DISCUSSIONS

In the following sections, the results of the two-stage (TS) cultivation of both strains are first described, followed by the results of the multi-stage (MS) cultivation, and finally the comparison between the two modes.

Effects on the Growth Rate

In the TS mode, C. vulgaris exhibited a typical growth curve (Figure 1) in the first stage (C₉₀ treatment), starting at nitrogen-rich conditions (90 mg.L^-1), increasing the cellular density up to 17.73×10⁶ cells.mL^-1. In the second stage, after a 50% v/v dilution, and starting at 30, 20, and 10 mg.L^-1 of N-NH₄ (C₃₀, C₂₀, and C₁₀treatments, respectively), the exponential phase of growth was maintained, but at lower specific growth-rates.

Figure 1
Growth curves (continuous lines) and nitrogen concentration curves (dotted lines) of S.obliquus and C. vulgaris grown in the TS mode.

A Tukey test showed that the three nitrogen-limited treatments are significantly different (p ≥0.05) from each other, in the maximum cell density achieved and the specific growth rate (Table 2). Although it can be argued that growth limitation was not achieved in this case, the reduced specific growth-rates indicate that the reduction in the available nitrogen did affect the metabolic paths of the culture.

In the case of S. obliquus, the nitrogen-rich treatment reached a maximum cell density of 19.9×10⁶ cells.mL^-1. However, after 12 h into the second stage, the culture entered the stationary phase for all reduced-nitrogen treatments (Figure 1 and Table 2); also, nitrogen uptake continued during the stationary phase, indicating that nitrogen limitation was achieved. Even when S. obliquus growth stopped after 12 h into the second stage, the specific growth rate in that period was slower in the C₂₀ and C₁₀ treatments than in C₉₀. Similar results were reported in microalgae cultures, where the effect of reducing nitrogen through medium dilution caused a reduction in the maximum cell density and the specific growth, leading to accumulation of lipids (Pravenkumar et al., 2012;. Mujtaba et al., 2012; Blair et al., 2014). One reason for this behavior is that certain nutrients (including N, P and Fe ions) are essential for cellular growth and structure synthesis; when these nutrients are present, cellular growth is preferred over lipid synthesis (Ho et al., 2010). However, nitrogen scarcity decreases the synthesis rate of cellular structures (including proteins and nucleic acids), whereas the synthesis rate of carbohydrates and lipids remains steady (Feng et al., 2011; Richardson et al., 1969).

Table 2
Maximum cell density and specific growth rates µ in the two-stage mode.

		C. vulgaris		S. obliquus
ID	Initial N-NH4 concentration (mg L-1)	Max. cell density (cells×106mL-1)	µ (d-1)	Max. cell density (cells×106mL-1)	µ (d-1)
C90	90	17.73 ± 0.05a	0.694 ± 0.007a	19.90 ± 0.05a	0.638 ± 0.014a
C30	30	13.82 ± 0.14b	0.455 ± 0.008b	14.50 ± 0.05b	0.600†± 0.035a
C20	20	12.22 ± 0.25c	0.321 ± 0.018c	9.17 ± 0.06c	0.247†± 0.024b
C10	10	13.29 ± 0.07d	0.397 ± 0.008d	11.93 ± 0.22bc	0.192†± 0.015b

• Different letters in the same column represent significant differences according to Tukey's test (p ≥ 0.05, n = 3), (± Standard deviation).
• ^†
  Calculated using only two measurements (0 and 12 h). After 12 h the culture reached stationary or death phase.

Low concentrations of nitrogen in the medium are associated with low nitrogen-uptake rates per cell. From the data in Table 3, it can be observed that, during the reduced nitrogen stage, C. vulgaris consumed ca. 50% of the medium nitrogen, and S. obliquus reached a maximum removal percentage of up to 74.2%, suggesting that, even after a sudden reduction of the nitrogen in the medium, both microalgae preserve the potential for nitrogen removal.

Table 3
Nitrogen removal, lipid content w, and lipid productivity P_L in the TS mode.

Species	Treatment ID	N-NH4 removal (%)	w (%w/w)	PL (mg L-1 d-1)	PL increment† (%)
C. vulgaris	C90	92.96	42.8 ± 6.1a	67.24
	C30	49.96	52.0 ± 5.7b	96.17	43.0
	C20	52.84	67.8 ± 1.2c	194.92	189.9
	C10	53.31	23.9 ± 2.8d	3.00	-95.5
S. obliquus	C90	85.33	37.4 ± 3.2a	79.67
	C30	62.90	60.0 ± 1.4b	133.48	67.5
	C20	70.24	63.4 ± 3.3b	106.92	34.2
	C10	74.21	24.4 ± 4.6c	6.82	-91.4

• ^*Different letters in the same column and for each species represent significant differences according to Tukey's test (p ≥ 0.05); (± Standard deviation)
• ^†
  Increment with respect to C₉₀

The results of MS cultivation of C. vulgaris and S. obliquus are shown in Figure 2. The maximum cell densities and specific growth rates for this mode of cultivation are shown in Table 4. As expected, the specific growth-rate in both species was higher during the initial nitrogen-rich stage (C₉₀), with values of 0.551 d^-1 and 0.837 d^-1 for C. vulgaris and S. obliquus, respectively, and decreased in the subsequent stages (C₆₀, C₄₀, C₂₀), reacting to the lower nitrogen concentrations in the medium.

Figure 2
Growth curves (continuous lines) and nitrogen concentration curves (dotted lines) of S. obliquusand C. vulgaris grown in the MS mode.

Table 4
Maximum cell density and specific growth rates in the MS mode.

		C. vulgaris		S. obliquus
Treatment ID	Initial N-NH4 concentration (mg L-1)	Max. cell density (cells×106mL-1)*	µ (d-1)	Max. cell density (cells×106ml-1)*	µ (d-1)
C90	90	22.48 ± 0.53a	0.551 ± 0.010a	17.27 ± 0.75a	0.837 ± 0.027a
C30	60	32.75 ± 0.55b	0.270 ± 0.004b	18.97 ± 0.15b	0.360 ± 0.062b
C20	40	21.63 ± 0.31a	0.134 ± 0.044c	15.68 ± 1.83ab	0.377 ± 0.127b
C10	20	12.17 ± 0.15c	0.124 ± 0.013c	14.76 ± 1.65ab	0.324 ± 0.223b

• ^*
  Different letters in the same column represent significant differences according to Tukey's test (p ≥ 0.05); (± Standard deviation)

The maximum cell density of C. vulgaris remained constant or even increased throughout the MS experiment until the last stage, where it reached a minimum value (Table 4). However, the specific growth-rate did decrease during the first three stages. A similar trend was observed for S. obliquus cultures, where the maximum density was achieved in the C₆₀ treatment and the specific growth-rate decreased after the second stage. These results show that both species showed a good adaptability to the gradual nitrogen-limitation, since growth did not stop but only slowed down.

It is also noteworthy that in TS cultivation, S. obliquus could not adapt to the sudden nitrogen scarcity and almost immediately entered the stationary phase. However, in the MS experiments, the gradual changes in the nitrogen concentrations allowed the microalgae to remain in the exponential phase of growth, with no significant changes in the specific growth rate after the second stage. These results suggest that the growth of both species was limited due to N-reduction in the culture medium, although the effect was stronger in C. vulgaris. Measurements of nitrogen removal support this conclusion (see data in Table 5), where the larger percentages of N-removal match the larger values of specific growth rates and vice versa.

Table 5
Nitrogen removal, lipid content w, and lipid productivity P_L in the MS mode.

Species	Treatment ID	N-NH4 removal (%)	w (%w/w)	PL (mg L-1 d-1)	PL increment† (%)
C. vulgaris	C90	86.31	53.2 ± 3.0a	79.19
	C60	80.40	51.1 ± 2.9a	53.46	-32.5
	C40	86.00	54.3 ± 2.8a	62.77	-20.7
	C20	74.83	62.9 ± 3.0b	108.01	36.4
S. obliquus	C90	87.63	37.6 ± 1.1a	60.20
	C60	82.43	40.1 ± 3.5a	77.74	29.1
	C40	73.63	32.4 ± 2.8ab	40.06	-33.5
	C20	56.12	28.4 ± 5.9b	-19.71	-132.7

• *Different letters in the same column and for each species represent significant differences according to Tukey's test (p ≥ 0.05); (± Standard deviation)
• ^†
  Increment with respect to C₉₀

Similar observations were reported by Mutlu et al. (2011), suggesting that nitrogen-limitation in microalgae cultures causes a decrease in cell density and chlorophyll a content, and accumulation of storage lipids. Also, Kim et al. (2007) report that Scenedesmus sp. exhibited growth inhibition due to the reduction of nitrogen and phosphorous, required by photosynthesis.

Effects on Lipid Content and Productivity

When certain types of stress are induced in the culture, many algae alter their lipid biosynthetic pathways towards the formation and accumulation of fatty acids, which do not perform a structural role, but instead serve primarily as a storage form of carbon and energy (Lin and Lin, 2011). It is generally accepted that stress by nitrogen-limitation inhibits cell division, without an immediate collapse of lipid production (Widjaja et al., 2009). In this work, this is well illustrated by the behavior of S. obliquus in TS cultivation, in which switching from nitrogen-rich (C₉₀) to nitrogen-limited conditions (C₃₀ and C₂₀) caused an increase in lipid content from 37.4% to 60.0% and 63.4% w/w, respectively (Table 3). These values are considerably higher than the 17.7% reported by Gouveia and Oliveira (2009) for the same species in nutrient-sufficient cultures, and similar to those found for S. obliquus by Xin et al.(2010), of 30% and 50% in cultures with nitrogen and phosphorous limitation, respectively.

The same trend was observed in TS cultivation of C. vulgaris, increasing w from 42.8% to 52.0% and 67.8% when switching from C₉₀ to C₃₀ and C₂₀, respectively. These values are considerably higher than those reported by Illman et al. (2000) for C. vulgaris grown in nutrient-sufficient cultures, ranging from 14 to 30%, and slightly lower than those reported by Rodolfi et al. (2009) of 70% w/w under nitrogen-limitation.

In the TS treatments with the largest reduction of nitrogen (C₉₀ to C₁₀), the lipid content of both species notoriously decreased. In these cases, C. vulgaris remained in the exponential phase of growth, and S. obliquus immediately entered the stationary phase. These results suggest that the sudden reduction to 10 mg.L^-1of N-NH₄ in the second stage does not allow either species to adjust their metabolism towards lipid storage and growth, which is the result of low adaptability to large nitrogen variations. Xin et al. (2010) reported that limiting nitrogen (2.5 mg.L^-1) and phosphorous (0.1 mg.L^-1) for Scenedesmus sp. LX1 caused a considerable increase of the lipid content (30 and 53%, respectively), but the net lipid-productivity is abated by a dominating decrease of the growth rate. Similar conclusions were drawn from the Report of Sheehan et al. (1998). However, in their extensive review, Williams and Laurens (2010) identified several works where nitrogen and phosphorous limitation resulted in a net increase of lipid productivity, despite the reduction in growth rates. The results from this work (Table 3) also support this observation: lipid productivities P_L in both species increased when switching from C₉₀ to C₃₀ and C₂₀, where C. vulgaris achieved the highest productivity in the C₂₀ batch (194.9 mg.L^-1.d^-1), and S. obliquus in the C₃₀ batch (133.5 mg.L^-1.d^-1). The increase in productivity is associated with lipid content increasing and the growth rate decreasing only moderately. The observed maximum values of P_Lare higher than most reported in the literature for photoautotrophic cultures (Table 1), and the trend of increasing P_L as the culture undergoes nitrogen-limitation confirms the trends observed by several previous investigations (Widjaja et al., 2009; Griffiths et al., 2011; Gouveia and Oliveira, 2009; Ho et al., 2010).

Table 5 shows the results for nitrogen removal, lipid content, and lipid productivity for the MS experiments for both species. In this mode of cultivation, the effect on lipid content and productivities was different for C. vulgaris and S. obliquus, supporting the assertion of Griffiths et al. (2011) that the response to nitrogen-limitation is species-specific.

For C. vulgaris, the second and third stages (C₆₀ and C₄₀) did not change the lipid content significantly, and the lipid productivity was reduced from 79.2 to 53.46 mg.L^-1.d^-1 due to the lower growth rate. However, in the fourth stage (C₂₀), both lipid content and productivity increased, achieving a maximum value of 108 mg.L^-1.d^-1, a 36.4% increase relative to the lipid productivity in the first stage. This result is in line with the observations of Widjaja et al. (2009), where C. vulgaris presented a decrease in lipid productivity after seven days under nitrogen-starvation, but extending the culture for 17 days resulted in a higher lipid productivity. This suggests that C. vulgaris needs more than five days of growing under nitrogen-limited conditions to redirect its metabolism towards a significant increase in lipid productivity.

S. obliquus behaved differently in MS cultivation. While in the second and third stages the lipid content did not change significantly, the lipid productivity increased slightly from 60.2 to 77.7 mg.L^-1.d^-1 (Table 5), due to the larger biomass concentration at the end of the second stage. From there, subsequent stages caused a decrease in both lipid content and productivity. From the growth curves of S. obliquus under MS cultivation (Figure 2) and the corresponding values of the specific growth rate in Table 4, it is evident that the culture was not greatly affected by the changes of the nitrogen concentration in the cultures, with the exception of the second stage (C₆₀). Thus, S. obliquus was able to maintain a steady growth in the exponential phase through the sequential stages, but lipid accumulation by nitrogen-limitation was not observed for this species in the MS mode. This suggests that, under a gradual reduction of nitrogen, S. obliquus can use the available nitrogen preferably for cell maintenance, influencing negatively in the lipid accumulation and productivity.

FAMEs Profiles of Microalgal Lipids

When the purpose of lipid production in microalgae cultures is the production of biodiesel, it is important to characterize the TAG fraction for the quality of the resulting biodiesel. Both cultivation time and nitrogen limitation affect microalgal lipids, not only increasing the lipid content, but also changing their composition (Widjaja et al., 2009). Thomas et al. (1984) analyzed the fatty acid composition of the lipids from seven species of fresh-water microalgae, and observed that all accumulated C_14:0, C_16:0, C_18:0, C_18:2, and C_18:3, that is, a majority of saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA). Hence, for the purpose of biodiesel production it is important to evaluate the changes in the fatty acid profile that may arise from the variations in the cultivation methods.

Tables 6a and 6b summarize the FAMEs profile found in C. vulgaris and S. obliquus, respectively, under the two cultivation modes studied in this work.

Table 6a
Profile of the fatty acids of C. vulgaris lipids (expressed as % of total fatty acids^*).

TS†				MS
FAMES	C90	C30	C20	C90	C60	C40	C20
C4:0	12.53(0.38)	15.04(1.33)	11.80 (0.59)	9.11 (0.30)	11.24 (0.64)	12.05 (3.25)	13.12 (0.94)
C14:0	10.27 (0.31)	11.36 (0.28)	10.84 (0.34)	8.45 (1.22)	5.47 (0.01)	6.10 (0.27)	6.18 (0.1)
C15:1	n.d.	n.d.	n.d.	8.88 (0.11)	8.03 (0.11)	6.24 (0.06)	6.87 (0.17)
C16:0	7.03 (0.11)	n.d.	7.05 (0.32)	5.12 (0.07)	4.77 (1.09)	3.93 (0.00)	4.15 (0.06)
C16:1	9.75 (0.42)	11.36 (0.28)	9.60 (0.27)	7.07 (0.29)	5.86 (0.13)	6.40 (0.11)	6.26 (0.00)
C17:0	n.d.	n.d.	n.d.	9.13 (0.30)	7.86 (0.04)	6.48 (0.01)	6.97 (0.19)
C18:1n9t	n.d.	n.d.	n.d.	3.99 (0.00)	3.60 (0.04)	6.65 (0.87)	4.93 (0.41)
C20:0	9.48 (0.04)	12.32 (0.17)	10.65 (0.07)	6.85 (0.02)	7.18 (0.22)	8.30 (0.38)	8.33 (0.04)
C18:3n6	n.d.	n.d.	n.d.	n.d.	5.48 (0.09)	4.75 (0.15)	4.52 (0.07)
C20:1	20.79 (0.12)	20.75 (0.12)	20.23 (0.15)	15.08 (0.10)	15.29 (0.46)	14.48 (0.58)	13.73 (0.05)
C21:0	7.17 (0.17)	7.04 (1.26)	7.31 (0.46)	5.20 (0.12)	5.23 (0.18)	4.78 (0.03)	4.87 (0.20)
C22:0	22.97 (0.56)	22.12 (0.58)	22.52 (1.10)	15.64 (0.42)	14.46 (0.36)	15.33 (0.86)	15.20 (0.37)
C22:1n9	n.d.	n.d.	n.d.	5.48 (0.28)	5.52 (0.10)	4.51 (0.03)	4.87 (0.24)
SFA	69.46(0.31)a	67.90(0.40)a	70.16(0.13)b	59.50(0.02)a	56.22(0.94)a	56.98(1.69)a	58.82(0.01)a
MUFA	30.54(0.34)a	32.11(0.40)a	29.83(0.12)b	49.50(0.02)a	38.30(0.85)a	38.27(1.54)a	36.66(0.05)a
PUFA	n.d.	n.d.	n.d.	n.d.	5.41(0.09)a	4.75(0.15)a	4.56(0.07)a
SFA:MUFA	2.27 a	2.11 a	2.35 b	1.47 a	1.47 a	1.49 a	1.60 a

• ^*
  Weight percentages, standard deviation between brackets
• ^†
  Lipid samples in the C₁₀ treatment were smaller than the detection limits.
• Different letters in the same row represent significant differences according to Tukey's test (p ≥ 0.05); (± Standard deviation)
• n.d. = not detected.

Table 6b
Profile of the FAMEs of S. obliquus lipids (expressed as % of total fatty acids^*).

TS†				MS
FAMES	C90	C30	C20	C90	C60	C40	C20
C4:0	12.28 (1.67)	23.85 (1.51)	18.43 (1.55)	11.55 (0.30)	15.50 (0.35)	26.13 (0.54)	21.81 (3.6)
C14:0	11.43 (0.16)	14.74 (1.54)	15.39 (1.62)	9.75 (0.04)	7.72 (0.07)	7.33 (0.06)	7.98 (0.62)
C16:1	10.69 (0.16)	8.54 (0.5)	10.53 (0.26)	9.12 (0.00)	7.61 (0.20)	7.06 (0.01)	6.91 (0.66)
C18:1n9t	n.d.	n.d.	n.d.	4.13 (0.04)	5.10 (0.68)	1.64 (1.08)	5.50 (3.51)
C20:0	14.20 (0.21)	9.19 (0.44)	11.38 (0.31)	12.11 (0.36)	9.59 (0.10)	8.54 (0.04)	8.14 (0.18)
C20:1	20.93 (0.28)	17.71 (2.52)	19.19 (0.90)	17.88 (0.08)	20.73 (0.78)	18.51 (0.07)	17.96 (0.06)
C21:0	n.d.	n.d.	n.d.	7.72 (0.56)	7.39 (0.14)	7.35 (0.01)	6.98 (0.14)
C22:0	20.96 (0.42)	14.09 (0.98)	14.68 (0.42)	17.90 (0.64)	17.32 (0.88)	15.03 (0.05)	16.53 (0.97)
C22:1n9	9.52 (1.64)	11.88 (2.50)	10.40 (1.83)	9.83 (0.90)	9.03 (0.31)	8.42 (0.43)	8.19 (0.47)
SFA	58.87 (1.19)a	61.87 (0.51)ab	59.88 (0.67)b	59.03(0.78)a	57.52(0.41)a	64.37(0.59)b	61.44 (2.32)ab
MUFA	41.13 (1.20)a	38.13 (0.50)b	40.12 (0.66)ab	40.96(0.80)a	42.48(0.42)a	35.62(0.60)b	39.01(2.30)ab
SFA:MUFA	1.43a	1.62b	1.49a	1.44a	1.35a	1.80b	1.60ab

• ^*
  Weight percentages, standard deviation between brackets
• ^†
  Lipid samples in the C₁₀ treatment were smaller than the detection limits.
• Different letters in the same row represent significant differences according to Tukey's test (p ≥ 0.05); (± Standard deviation)
• n.d. = not detected.

It can be observed that the lipids content of C. vulgaris is mainly SFA and monounsaturated fatty-acids (MUFA). The profiles show that the ratio of SFAs to MUFAs did not change significantly after reducing nitrogen in the medium, with the exception of the C₂₀ treatment, where the production of SFA was slightly favored. Unfortunately, the C10 treatments did not yield enough lipids for the GC measurements. On the other hand, cultures of C. vulgaris in the MS mode did not exhibit significant changes in the lipid composition throughout the sequential stages, and the overall ratio SFA:MUFA remained constant throughout the sequential batches.

The cultivation mode did affect the FAMEs profile. In the MS mode, the PUFA C_18:3 was detected for C. vulgaris, and the relative compositions of most FAMEs in both species were different than in the TS mode, reaffirming the conclusion by Damiani et al. (2010) that the growth conditions affect the FAMEs profile. Similar results were reported by Makulla (2000) for the microalga S. obliquus, noting that an increase in the dilution rate caused the SFA fraction to increase from 44.97% in a culture with no dilution to 50.73% when cultivated with a dilution rate of 0.48 d^-1. In addition, studies by Lin and Lin (2011) showed that the C_18:3 fraction decreased dramatically during the nitrogen starvation phase. Finally, Johnson and Wen (2009) found that the C₁₆ and C₁₈ series of Chlorella sp. changed significantly during 5 days of culture.

It is also important to note that the main components of the lipid profiles were long-chain fatty acids (C_22:0 and C_20:1), and a large proportion of C_4:0. This would mean a large cetane number for the resulting biodiesel, although it may result in poor cold-flow properties. Also, the low level of MUFAs and PUFAs suggest that the resulting biodiesel would have good oxidative stability.

In S. obliquus lipids, only SFA and MUFA were identified. The main compounds in S. obliquus lipids were long-chain fatty acids (C_22:0 and C_20:1). Although C_4:0concentrations were high (>13% in all treatments), the overall composition was more balanced than in C. vulgaris lipids. In the TS mode, the sudden nitrogen limitation had the effect of increasing the composition of lower-sized carbon chains (C_4:0, C_14:0), increasing the fraction of SFA, and decreasing the MUFAs content, so that the ratio of SFA:MUFA varied from 1.4-1.6 (in C₉₀) to 2.1-2.3 in C₃₀ and C₂₀, respectively. On the other hand, the MS mode did not seem to affect noticeably the fraction of SFA or MUFA, and the ratio SFA:MUFA remained in the range of 1.3 to 1.8.

Similarly to C. vulgaris, the composition of S. obliquus lipids can lead to good biodiesel quality given a small percentage of MUFAs, virtually no presence of PUFAs, and long-chain fatty acids as main components. Although the latter factor may result in high viscosity, the resulting biodiesel may still be appropriate for concentrated fuel blends.

CONCLUSIONS

Both nitrogen-reduction strategies increased the net lipid-productivity and lipid content. The highest lipid-productivities occurred in TS cultivation (194.92 and 133.48 mg.L^-1.d^-1 for C. vulgaris and S. obliquus, respectively), and are among the highest values reported to date. C. vulgaris adapted well in the TS mode, but in MS cultivation the accumulation of lipids was delayed for at least 5 days. The microalgae

S. obliquus were not able to sustain growth in the TS mode, increasing their lipid content; however, in the MS mode the microalgae adapted well to the reduced nitrogen concentrations and the lipid-accumulation effect was not observed. Both species presented a good potential for nitrogen removal from the artificial wastewater medium, and in general, the removal efficiencies were larger in MS cultivation. The fatty-acid profiles of the lipids from both species showed good potential for biodiesel production in terms of their content of saturated, monounsaturated, and polyunsaturated fatty-acids.

ACKNOWLEDGEMENTS

This research was supported by the Mexican Council of Science and Technology (CONACyT) project 145521, and the Secretariat of Public Education of Mexico (SEP) project PROMEP 103.5/09/7341.

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Publication Dates

• Publication in this collection
  Apr-Jun 2015

History

• Received
  17 Feb 2014
• Reviewed
  06 July 2014
• Accepted
  22 July 2014