Bioresource Technology 152 (2014) 241–246
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Effect of phosphorus on biodiesel production from Scenedesmus obliquus under nitrogen-deficiency stress Fei-Fei Chu a, Pei-Na Chu b, Xiao-Fei Shen a, Paul K.S. Lam a,c, Raymond J. Zeng a,b,⇑ a
Advanced Laboratory for Environmental Research and Technology, USTC-CityU, Suzhou, PR China Department of Chemistry, University of Science and Technology of China, Hefei 230026, PR China c State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Nitrogen starvation enhanced
biodiesel productivity compared to nitrogen repletion. Biodiesel productivity was further strengthened by the supply level of phosphorus. More P was accumulated in cells under nitrogen starvation with sufficient P supply. A potential application to combine P removal with biodiesel production via microalgae.
a r t i c l e
i n f o
Article history: Received 20 August 2013 Received in revised form 29 October 2013 Accepted 7 November 2013 Available online 14 November 2013 Keywords: Scenedesmus obliquus FACHB-417 Nitrogen deficiency Phosphorus effect Biodiesel productivity Lipid trigger
a b s t r a c t In order to study the effect of phosphorus on biodiesel production from Scenedesmus obliquus especially under nitrogen deficiency conditions, six types of media with combinations of nitrogen repletion/depletion and phosphorus repletion/limitation/depletion were investigated in this study. It was found that nitrogen starvation compared to nitrogen repletion enhanced biodiesel productivity. Moreover, biodiesel productivity was further strengthened by varying the supply level of phosphorus from depletion, limitation, through to repletion. The maximum FAMEs productivity of 24.2 mg/L/day was obtained in nitrogen depletion with phosphorus repletion, which was two times higher than that in nutrient complete medium. More phosphorus was accumulated in cells under the nitrogen starvation with sufficient phosphorus condition, but no polyphosphate was formed. This study indicated that nitrogen starvation plus sufficient P supply might be the real ‘‘lipid trigger’’. Furthermore, results of the current study suggest a potential application for utilizing microalgae to combine phosphorus removal from wastewater with biodiesel production. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Renewable energy regarding biodiesel from microalgae has already been studied for decades (Wijffels and Barbosa, 2010). Various strategies have been used to increase algal biomass yield and/or lipid content in order to reduce the cost of biodiesel production. These strategies include nutrient limitation or starvation, ⇑ Corresponding author at: Department of Chemistry, University of Science and Technology of China, Hefei 230026, PR China. Tel./fax: +86 551 63600203. E-mail address: [email protected] (R.J. Zeng). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.11.013
high-intensity illumination, heterotrophic cultivation, etc. (Liu et al., 2008; Miao and Wu, 2006; Yeesang and Cheirsilp, 2011). Among these, nitrogen starvation is deemed as a major ‘‘lipid trigger’’ in green algae species (Sheehan et al., 1998). However, during subsequent decades, many high lipid-accumulating microalgal strains have been found to be associated with slow growth rate under nitrogen limited conditions, which leads to little improvement or even a decrease in overall oil productivity (El-Sheekh et al., 2013; Gordillo et al., 1998; Zhila et al., 2005). Some researchers believe that algal cultivation under sufficient nitrogen conditions is
242
F.-F. Chu et al. / Bioresource Technology 152 (2014) 241–246
more economical for lipid production than under deficient nitrogen conditions (Griffiths and Harrison, 2009; Mujtaba et al., 2012; Wijffels and Barbosa, 2010). Interestingly, our previous study found that phosphorus played an important role in enhancing lipid productivity of Chlorella vulgaris under nitrogen deficient conditions. With sufficient phosphorus supply, lipid productivity under nitrogen deficient conditions was 1.35 times higher than that of nutrient complete media. Moreover, phosphorus was luxuriously uptaken and accumulated as polyphosphate (Poly-P) (Chu et al., 2013). Actually, the wide distribution of Poly-P in many microorganisms, including blue–green algae and its accumulation under nutritional imbalance conditions, have been known for several decades (Harold, 1966). However, most researchers using stimulation method of nitrogen starvation for lipid production usually ignore the role of phosphorus (P), and this is one possible reason for the less than satisfactory lipid productivity under nitrogen depletion conditions (Rodolfi et al., 2009; Widjaja et al., 2009). It is unclear whether it is universally applicable for other microalgae that sufficient phosphorus supply over and above what is needed for growth is necessary in nitrogen deficiency media for higher lipid productivity. Meanwhile, phosphorus morphology in algal cells also needs to be investigated. Among microalgae, Scenedesmus obliquus contains oleic, palmitic and stearic acids as main fatty acids which are preferable for biodiesel production, and is also an excellent candidate for the economic combination of wastewater treatment and biodiesel production (Abd El Baky et al., 2012; Gouveia and Oliveira, 2009). Therefore, many researchers select S. obliquus as a potential and excellent source for lipid production. It has been found that the starvation or the limitation of nitrogen and/or phosphate could improve lipid content. However, large variations in lipid productivity exists amongst various studies perhaps because different experimental conditions and operational methods were applied (Abd El Baky et al., 2012; El-Sheekh et al., 2013; Ho et al., 2012; Mallick, 2009). For example, fatty acid content was enhanced by 54% over control under nitrogen deficiency, but its productivity was decreased due to the growth limitation (El-Sheekh et al., 2013). Li et al. (2010) reported that the lipid content of S. obliquus was the highest under nitrogen or phosphorus limitation, however, the highest lipid productivity was not achieved simultaneously. On the contrary, Breuer et al. (2012) found that S. obliquus showed the highest triacylglycerol (TAG) productivity among nine common strains under nitrogen starvation conditions, and the value was surprisingly 5.29 times that of nitrogen sufficient conditions. However, none of the researchers considered the effect of phosphorus under nitrogen starvation conditions. The aim of this study was to evaluate the role of P in biodiesel production from S. obliquus under nitrogen deficient conditions. Specifically, three levels of phosphate (sufficient, limited and deficient) were adopted under nitrogen sufficient and deficient conditions, respectively. The profiles of algal growth and nutrient absorption were monitored, and forms of phosphorus assimilated in cells were investigated. It was anticipated that P might play a role in enhancing the application of nitrogen deficiency or limitation for biodiesel production.
2. Methods 2.1. Strains and culture conditions S. obliquus FACHB-417 was obtained from Freshwater Algae Culture Collection of the Institute of Hydrobiology (FACHB), Chinese Academy of Sciences, Hubei province, China. The algae cultivation device was designed as described in a previous study (Chu et al., 2013). BG11 medium was autoclaved at 121 °C for 30 min and then
used to cultivate S. obliquus FACHB-417. The cultivation was maintained at 25 ± 2 °C under a light intensity of 120 lmol photons/ m2 s with photoperiodicity 16:8 (light for 16 h and dark for 8 h). Mixed gases (4% CO2 in air) were bubbled to the medium through a 0.20 lm PTFE membrane (SRP65, Sartorius, Germany) at a flow rate of 400 mL/min. The solution pH was kept at 6–8. 2.2. Experimental setup In order to investigate the effect of phosphorus on biomass and biodiesel production from S. obliquus, especially under nitrogen deficiency conditions, six types of media were adopted in this study, which included: sufficient nitrogen and sufficient phosphorus (N&P), sufficient nitrogen and phosphorus limited (N&P-lim), sufficient nitrogen and phosphorus deficiency (N&P-), nitrogen deficiency and sufficient phosphorus (N-&P), nitrogen deficiency and phosphorus limited (N-&P-lim), simultaneous nitrogen and phosphorus deficiency (N-&P-). S. obliquus FACHB-417 strain was pre-cultivated for 6 days and then centrifuged at 6000 rpm for 5 min. After washing with BG11 medium (without N and P) twice to remove residual orthophosphate and nitrate nitrogen, cells were suspended at an initial concentration of 160 mg/L in each of the six types of media. The concentration of phosphorus PO3 4 -P in the culture of N&P and N-&P experiments was 45 mg/L. Moreover, approximately 3.5 mg/L phosphorus was added on day 6 to induce a phosphorus limited condition for N&P-lim and N-&P-lim. The initial concentrations of nitrate and orthophosphate for these six media are shown in Table 1. The experiments for each type of medium lasted 16 days and operated in two duplicates. The liquid sample was collected with respect to time to determine biomass production, residual nitrate and orthophosphate concentrations. Moreover, algal cells were collected intermittently after centrifugation and then freeze-dried using a vacuum freeze dryer (HETO power Dry Pl3000, USA) to obtain algal powder. Subsequently, protein/carbohydrate/FAMEs contents and P content per 107 cells were measured. 2.3. Analytical methods 2.3.1. Determination of growth and nutrient consumption profiles Biomass sample of broth (10 mL) was filtered through a cellulose acetate membrane filter and then dried at 105 °C overnight. The dry weight of microalgae was obtained by the difference between the dry weight of the membrane blank and the loaded filter. Cell counting was conducted using an improved Neubauer haemocytometer slide under an inverted biological phase contrast microscope (Meiji TC5300, Japan). Algal powder (5 mg) was resuspended in distilled water and then digested using 5% potassium persulfate solution. Subsequently, the total phosphate contents were determined after all types of phosphorus were converted into ortho3 phosphate. The measurements of NO 3 -N; PO4 -P were conducted using a water quality autoanalyzer (Aquakem 200, ThermoFisher, Finland) according to standard methods (APHA, 1998). The assim3 ilation rates of NO 3 -N and PO4 -P were calculated using the linear regression slopes of the data with time. 2.3.2. Determination of starch content Freeze-dried algae powder (3 mg) was suspended in 2.5 mL of distilled water and autoclaved for 1 h at 135 °C for starch solubilization. Total starch in algal cells was analyzed using a commercial enzymatic Starch Assay Kit (SA-20, Sigma–Aldrich) based on amyloglucosidase digestion which converts starch to glucose. 2.3.3. Determination of crude protein content The crude protein content in algal cells was determined using the indirect method reported by Becker (1993). A correlation of
243
F.-F. Chu et al. / Bioresource Technology 152 (2014) 241–246 Table 1 The initial concentrations of nitrate and orthophosphate for six types of culture media. Initial concentration (mg/L) N NO 3 -N P PO3 4 -P a
N&P
N&P-lim
N&P-
N-&P
240
240
240
0
45
3.0a
0
45
N-&P-lim
N-&P-
0
0
3.0a
0
Approximately 3.5 mg/L phosphorus was added again on day 6 to induce a phosphorus limited condition for N&P-lim and N-&P-lim.
‘‘protein content = nitrogen content 6.25’’ was used to describe the relationship between protein content and nitrogen content. The nitrogen content of algal cells was measured using an elemental analyzer (Elementar Vario MACEO, Germany). 2.3.4. Determination of biodiesel production and 31P NMR Biodiesel was determined as fatty acid methyl esters (FAMEs) after direct transesterification adapted from Rodriguez-Ruiz et al. (1998). Lyophilized algal powder (20 mg) was collected and then freshly prepared transesterification reagent (a mixture of acetyl chloride and methanol (1:9 v/v), 2 mL), was added. Also internal standard methyl benzoate (0.5 g/L in hexane) was added after transesterification and the samples were analyzed using a gas chromatograph (Agilent 6890, CA). All the detailed steps and operating conditions are described in our previous study (Chu et al., 2013). The total FAMEs content, which is representative the oil content in the cells, was calculated as the percentage of dry biomass. FAMEs productivity was calculated based on Eq. (1):
FAMEs productivity ðmg=L=dayÞ Biomass production ðmgÞ FAMEs content ð%Þ ¼ Working volume ðLÞ Cultivation time ðdayÞ
ð1Þ
For the measurement of phosphorus forms in algal cells after 16-day cultivation, the samples were prepared using the method of perchloric acid extraction and then measured using 31P NMR. The details are given in our previous study (Chu et al., 2013). 2.4. Statistical analysis Statistical analysis was performed using Excel 2007 software (Microsoft, USA). Variables were reported as significant at 95% confidence (p-value less than or equal to 0.05). 3. Results and discussion 3.1. Algal growth profile Fig. 1 shows the growth profiles of S. obliquus FACHB-417 in six types of media. During the 16-day experiment, the growth rates of N&P, N&P-lim and N&P- were 104.0, 95.9 and 42.1 mg/L/day, respectively, while the rates for N-&P, N-&P-lim and N-&P- were 57.1, 52.4, 34.6 mg/L/day, respectively. To determine whether the effects were significant, p-values for the two variables (N supply and P concentration) were obtained after statistical analysis. Nitrogen supply (p = 3.3 106 < 0.05) and ortho-phosphate concentration (p = 8.63 106 < 0.05) were found to have significant effects on biomass growth. Significant interactions occurred between nitrogen supply and P concentration with p-value of 0.0005 (Fig. 1). The rates under nitrogen sufficient conditions were higher than the nitrogen starvation conditions with the same level of phosphorus supply. This phenomenon for S. obliquus was verified by other researchers as well. Mallick (2009) reported extremely little increase of biomass yield under conditions of N limitations or deficiencies, while El-Sheekh et al. (2013) found no growth of S. obliquus in the cultures devoid of nitrogen. Moreover, in both
Fig. 1. Profiles of biomass growth of S. obliquus FACHB-417 cultured in different types of media under the conditions of 4% CO2, 25 ± 2 °C and light intensity of 120 lmol photons/m2 s. Values shown are averages of two samples ± range.
nitrogen sufficient and starvation conditions, the rates of biomass increase under phosphorus sufficient conditions were slightly higher than under phosphorus limitation conditions, but were far greater than under phosphorus starvation conditions. These results indicated that phosphorus was vital for maintaining the normal metabolism of algal cells. 3.2. Nutrient assimilation The concentrations of nitrogen NO3 -N and phosphorus 3 PO4 -P in the media were determined and their assimilation profiles by S. obliquus are shown in Fig. 2. The concentration of NO 3 -N decreased rapidly from 240 mg/L to 144.7 mg/L in N&P and to 146.2 mg/L in N&P-lim, respectively, while it was almost unchanged in N&P- condition. Phosphorus concentration had a significant effect on nitrate assimilation (p = 0.001 < 0.05) (Fig. 2A). This phenomenon was also observed when C. vulgaris grew in phosphorus depletion media (Chu et al., 2013). It seems a very plausible hypothesis that the existence of phosphorus is essential for nitrate absorption by algal cells. Cellular energy transduction is closely associated with inorganic phosphate. Various aspects of metabolism including transportation of nitrate ion and proteins synthesis (nitrogen needed) would be down-regulated via adenylate limitation and plasmalemma H+-ATPase activity (Beardall et al., 2005). Moreover, the nitrate assimilation rate in N&P-lim almost equaled the rate in N&P (Table 2). It indicated that a small quantity of phosphorus was adequate to support the nitrogen assimilation. Fig. 2B illustrates the phosphorus assimilation capacity of S. obliquus FACHB-417 under different conditions. In N&P-lim and N-&P-lim, 3.5 mg/L phosphorus was added on day 6, and its concentration remained at a very low level again after two days of rapid consumption. As shown in Table 2, the overall phosphorus assimilation rate under N&P was 0.94 mg/L/day, which was even more than that in N-&P (0.78 mg/L/day), however, the nitrogen supply had no significant effect on the ortho-phosphate assimilation (p = 0.25 > 0.05) (Fig. 2B). This situation was quite different from C. vulgaris reported in our previous work (Chu et al., 2013).
244
F.-F. Chu et al. / Bioresource Technology 152 (2014) 241–246
Fig. 3. Phosphorus content per cell of S. obliquus FACHB-417 cultured with the media of N&P, N-&P, N&P-lim. Values shown are averages of two samples ± range.
Fig. 2. Profiles of nutrients assimilation by S. obliquus FACHB-417 cultured in different types of media under the conditions of 4% CO2, 25 ± 2 °C and light intensity 3 of 120 lmol photons/m2 s. (A) NO 3 -N. (B) PO4 -P. Values shown are averages of two samples ± range.
Table 2 Summary of the overall N and P consumption rates of six media during16-day cultivation. Rate (mg/L/day) N NO 3 -N P PO3 4 -P
N&P
N&P-lim
N&P-
N-&P
N-&P-lim
N-&P-
6.00
5.86
0.38
–
–
–
0.94
0.34
–
0.78
0.27
–
–, No data available.
A low phosphorus consumption rate of 0.49 mg/L/day was found in the control with complete nutrients while nitrogen starvation was associated with a strong phosphorus assimilation rate (1.87 mg/L/ day) in N-&P (Chu et al., 2013). 3.3. Phosphorus content in cell and
31
P NMR
S. obliquus FACHB-417 was investigated not only for its phosphorus absorption rate, but also for the phosphorus content and its form in cells. The phosphorus content inside the cells in N&P, N-&P and N&P-lim are shown in Fig. 3. After 16-day cultivation, phosphorus content gradually increased from 2.6 to 12.3 lg/ 107 cells under N-&P condition, stabilized at an average value of 5.4 lg/107 cells in N&P, but declined to 1.9 lg/107 cells in N&Plim. It demonstrated that nitrogen starvation provided a trigger for more phosphorus to be assimilated in S. obliquus cells in nitrogen starvation media, though phosphorus assimilation rates were quite similar between N&P and N-&P (Fig. 2B). The values of phosphorus content in these three conditions were significantly different (p = 0.0002 < 0.05) (Fig. 3). Li et al. (2010) also reported that the assimilation rate of TP was significantly enhanced as initial TN concentration decreased in the media.
Fig. S1 shows the 31P NMR spectra of S. obliquus under N&P and N-&P conditions. Inorganic orthophosphate (Pi) and organic phosphorus were observed distinctly in the two spectra. However, no Poly-P accumulation, which corresponded to the chemical shift mainly at 20.00 ppm, was found under both conditions. C. vulgaris showed Poly-P accumulation under nitrogen starved conditions (Chu et al., 2013). Inorganic polyphosphates have been found in organisms such as eubacteria, fungi, algae, protozoa and even in various tissues of higher plants and animals, and its accumulation was usually boosted under stress conditions including nitrogen depletion (Harold, 1966; Kulaev and Vagabov, 1983). Poly-P are thought to function not only as a phosphorus reserve, but also as an energy source reserve which enables cells to grow smoothly under stress conditions (Harold, 1966). However, inorganic polyphosphates are not obligatory cell components and may not be vitally important for organisms (Kulaev and Vagabov, 1983). It seems that Poly-P accumulation in microalgae is far from being universal. Meanwhile, more inorganic P was found in N-&P than in N&P, which was consistent with the trend of P contents in Fig. 3. 3.4. Composition of storage products Protein, starch and lipid are major storage products in microalgae. As shown in Fig. 4A, protein content reduced rapidly from 52.8% to an average value of 17.9% in the first 4 days under all nitrogen starvation conditions since nitrogen is a vital element for protein synthesis. Protein contents remained almost unchanged in N&P and N&P-lim but had a slight decline in N&P- which could be explained by the lack of metabolic energy attributed to phosphorus depletion. The starch level rose rapidly within 4 days and then slowly increased during the following 12 days, with final contents of 28.9%, 26.2% and 25.6% in N-&P, N-&P-lim and N-&P-, respectively. There was not much change (around 7%) under the conditions with sufficient nitrogen supply (Fig. 4B). In terms of FAMEs profile, accumulation lagged behind starch and increased dramatically after 4 days under all nitrogen depleted conditions, and the final contents of N-&P, N-&P-lim and N-&P- were 37.6%, 35.4% and 35.0%, respectively. A dramatic enhancement of lipid content from 10% to 43% had been observed by Mallick (2009) as well under N-deficient condition. Similar to starch, FAMEs content was almost unchanged under nitrogen sufficient conditions (Fig. 4C). Regarding the effect of P, it seemed to have a positive effect for starch and FAMEs as both had an estimated increase of 2% at the end of the cultivation process with sufficient P supply (N-&P) compared with limited P supply (N-&P-lim) or P depletion (N-&P-) (Fig. 4B and C). Statistical analysis indicated that nitrogen supply had a significant effect on protein, starch and FAMEs
F.-F. Chu et al. / Bioresource Technology 152 (2014) 241–246
245
Fig. 5. FAMEs productivity of S. obliquus FACHB-417 cultured with different types of media under the conditions of 4% CO2, 25 ± 2 °C and light intensity of 120 lmol photons/m2 s. Values shown are averages of two samples ± range.
Fig. 4. Storage products of S. obliquus FACHB-417 grown under different types of media under the conditions of 4% CO2, 25 ± 2 °C and light intensity of 120 lmol photons/m2 s. (A) Protein. (B) Starch and (C) total FAMEs. Values shown are averages of two samples ± range.
contents, while ortho-phosphate concentration had a significant effect on protein, but not on starch and FAMEs (Fig. 4). The lipid accumulation accompanied by starch increase was also observed in other algal species, such as S. obliquus, Chlorella zofingiensis, Phaeodactylum tricornutum and Neochloris oleoabundans, and all without exception showed that the accumulation of oil lagged behind that of starch (Breuer et al., 2012). This can be explained by an examination of biosynthesis metabolic pathways of starch and oil in Chlamydomonas reinhardtii that the biosynthesis of the two organic compounds competed not only for common carbon precursors, but also for metabolic energy and space in chloroplasts (Fan et al., 2012). 3.5. FAMEs productivity FAMEs productivity reflects the integration of biomass productivity and FAMEs content. As shown in Fig. 5, higher FAMEs
productivity was found under nitrogen starvation conditions than under nitrogen sufficient conditions with equal phosphorus content. Moreover, higher phosphorus supply resulted in higher FAMEs productivity under nitrogen deficiency (N-&P > N-&Plim > N-&P-). The highest FAMEs productivity was 24.2 mg/L/day in N-&P. In N-&P, FAMEs productivity was approximately two times greater compared to N&P during the whole cultivation period (Fig. 5). Nitrogen supply (p = 1.4 105 < 0.05) and orthophosphate concentration (p = 0.0003 < 0.05) were found to have significant effects on FAMEs productivity, however, no significant interactions occurred between these two variables (p = 0.25 > 0.05) (Fig. 5). Clearly, these results demonstrated that nitrogen starvation did enhance biodiesel productivity, which however contradicts most nitrogen deficient studies concerning S. obliquus (El-Sheekh et al., 2013; Li et al., 2010; Mallick, 2009). For example, El-Sheekh et al. (2013) reported that fatty acids productivity reduced sharply due to growth inhibition under nitrogen limitation despite the fact that a lipid content of 1.54 times greater was obtained. Phosphorus demonstrated a strong positive role in enhancing biodiesel productivity under both nitrogen depletion and sufficient conditions, and this observation was consistent with our previous study of C. vulgaris (Chu et al., 2013). Unfortunately, most studies in literature did not consider the importance of sufficient phosphorus supply for the enhancement of lipid productivity with nitrogen starvation stimulation. It is known that lipid synthesis in microalgae is via so-called CCMs (CO2 concentrating mechanisms), a pathway responsible for the acquisition and fixation of inorganic carbon from the environment, in which CO2 is assimilated and converted to organic compounds as lipid and carbohydrate. An enhancement of CCMs capacity in Dunaliella tertiolecta was observed under nitrogen stress, although the photosynthetic capacity was compromised (Young and Beardall, 2005). However, the CCMs activity in Chlorella emersonii was markedly down-regulated under phosphate limited conditions as it led to a slow growth rate and an increase of inorganic carbon (2005). Carbon acquisition is an ATP requiring process. It is likely that P limitation results in decreased ATP levels which reduce CCM activity, and finally decreases lipid productivity. 3.6. Implications of this work S. obliquus and C. vulgaris are two important and well studied green algae for lipid production. In our previous study on C. vulgaris (Chu et al., 2013), it was found that phosphorus uptake rates were 5.3 and 19.6 mg P/g biomass in N&P and N-&P, respectively, while
246
F.-F. Chu et al. / Bioresource Technology 152 (2014) 241–246
the rates for S. obliquus in this study were 9.1 and 13.0 mg P/g biomass, respectively. When comparing N-&P to N&P, lipid productivity was enhanced 1.35 times and 2 times for C. vulgaris and S. obliquus, respectively. Meanwhile, Poly-P was found in C. vulgaris, but not in S. obliquus. These findings indicated that the form of P inside the cells was not crucial for the enhancement of biodiesel production. In summary, both our studies on C. vulgaris and S. obliquus showed that P likely plays an important role in green algae in enhancing lipid productivity under nitrogen deficiency condition. Whether the role of P in the enhancement of biodiesel productivity can be extended to other types of algal species beyond green algae requires further investigation. Nevertheless, it indicates that nitrogen starvation plus sufficient P supply may be the real ‘‘lipid trigger’’ for microalgal biodiesel production. Furthermore, due to the faster P uptake rate under nitrogen starvation, it suggests a potential application for utilizing microalgae to combine phosphorus removal from wastewater with biodiesel production. For example, microalgae can grow normally in the first stage under nitrogen sufficient conditions, and then enter into the second stage with nitrogen deprivation and sufficient phosphorus to enhance biodiesel productivity and phosphorus removal. However, more investigations are required to verify this combined process, especially with regard to technical and economical aspects. 4. Conclusions This study demonstrated that nitrogen depletion was an effective way for stimulating biodiesel production from S. obliquus. Moreover, biodiesel productivity was further strengthened by altering the supply level of phosphorus from depletion, limitation, through to repletion. The highest FAMEs productivity of 24.2 mg/L/ day was obtained in N-&P, and the level was two times that of nutrient complete medium. Furthermore, more phosphorus was accumulated in cells under the condition of nitrogen starvation with sufficient phosphorus compared to nutrient complete medium, but no Poly-P formation was observed. This finding indicated that nitrogen starvation plus sufficient phosphorus supply might be the real ‘‘lipid trigger’’. Acknowledgements The authors would like to acknowledge the financial support of the Hundred-Talent Program of Chinese Academy of Sciences and the Program for Changjiang Scholars and Innovative Research Team in University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2013. 11.013. References Abd El Baky, H.H., El-Baroty, G.S., Bouaid, A., Martinez, M., Aracil, J., 2012. Enhancement of lipid accumulation in Scenedesmus obliquus by optimizing CO2 and Fe3+ levels for biodiesel production. Bioresour. Technol. 119, 429–432.
APHA, 1998. Standard Methods for Examination of Water and Wastewater, twentieth ed. American Public Health Association, Washington, DC. Beardall, J., Roberts, S., Raven, J.A., 2005. Regulation of inorganic carbon acquisition by phosphorus limitation in the green alga Chlorella emersonii. Can. J. Bot.-Rev. Canadienne De Botanique 83 (7), 859–864. Becker, E.W., 1993. Microalgae: Biotechnology and Microbiology. Cambridge University Press. Breuer, G., Lamers, P.P., Martens, D.E., Draaisma, R.B., Wijffels, R.H., 2012. The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains. Bioresour. Technol. 124, 217–226. Chu, F.-F., Chu, P.-N., Cai, P.-J., Li, W.-W., Lam, P.K.S., Zeng, R.J., 2013. Phosphorus plays an important role in enhancing biodiesel productivity of Chlorella vulgaris under nitrogen deficiency. Bioresour. Technol. 134, 341–346. El-Sheekh, M., Abomohra, A.E.-F., Hanelt, D., 2013. Optimization of biomass and fatty acid productivity of Scenedesmus obliquus as a promising microalga for biodiesel production. World J. Microbiol. Biotechnol. 29 (5), 915–922. Fan, J., Yan, C., Andre, C., Shanklin, J., Schwender, J., Xu, C., 2012. Oil accumulation is controlled by carbon precursor supply for fatty acid synthesis in Chlamydomonas reinhardtii. Plant Cell Physiol. 53 (8), 1380–1390. Gordillo, F.J.L., Goutx, M., Figueroa, F.L., Niell, F.X., 1998. Effects of light intensity, CO2 and nitrogen supply on lipid class composition of Dunaliella viridis. J. Appl. Phycol. 10 (2), 135–144. Gouveia, L., Oliveira, A.C., 2009. Microalgae as a raw material for biofuels production. J. Ind. Microbiol. Biotechnol. 36 (2), 269–274. Griffiths, M.J., Harrison, S.T.L., 2009. Lipid productivity as a key characteristic for choosing algal species for biodiesel production. J. Appl. Phycol. 21 (5), 493–507. Harold, F.M., 1966. Inorganic polyphosphates in biology: structure, metabolism, and function. Bacteriol. Rev. 30 (4), 772. Ho, S.-H., Chen, C.-Y., Chang, J.-S., 2012. Effect of light intensity and nitrogen starvation on CO2 fixation and lipid/carbohydrate production of an indigenous microalga Scenedesmus obliquus CNW-N. Bioresour. Technol. 113, 244–252. Kulaev, I.S., Vagabov, V.M., 1983. Polyphosphate metabolism in microorganisms. Adv. Microb. Physiol. 24, 83–171. Li, X., Hu, H.Y., Gan, K., Sun, Y.X., 2010. Effects of different nitrogen and phosphorus concentrations on the growth, nutrient uptake, and lipid accumulation of a freshwater microalga Scenedesmus sp.. Bioresour. Technol. 101 (14), 5494–5500. Liu, Z.Y., Wang, G.C., Zhou, B.C., 2008. Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresour. Technol. 99 (11), 4717–4722. Mallick, S.M.N., 2009. Microalga Scenedesmus obliquus as a potential source for biodiesel production. Appl. Microbiol. Biotechnol. 84 (2), 281–291. Miao, X., Wu, Q., 2006. Biodiesel production from heterotrophic microalgal oil. Bioresour. Technol. 97 (6), 841–846. Mujtaba, G., Choi, W., Lee, C.-G., Lee, K., 2012. Lipid production by Chlorella vulgaris after a shift from nutrient-rich to nitrogen starvation conditions. Bioresour. Technol. 123, 279–283. Rodolfi, L., Zittelli, G.C., Niccolo‘, Bassi, Padovani, G., 2009. Microalgae for Oil: Strain Selection, Induction of Lipid Synthesis and Outdoor Mass Cultivation in a LowCost Photobioreactor. Biotechnology and Bioengineering 102 (1), 100–112. Rodriguez-Ruiz, J., Belarbi, E.H., Sanchez, J.L.G., Alonso, D.L., 1998. Rapid simultaneous lipid extraction and transesterification for fatty acid analyses. Biotechnol. Tech. 12 (9), 689–691. Sheehan, J., Dunahay, T., Benemann, J., Roessler, P., 1998. A Look Back at the US Department of Energy’s Aquatic Species Program: Biodiesel from Algae. National Renewable Energy Laboratory Golden. Widjaja, A., Chien, C.C., Ju, Y.H., 2009. Study of increasing lipid production from fresh water microalgae Chlorella vulgaris. J. Taiwan Inst. Chem. Eng. 40 (1), 13– 20. Wijffels, R.H., Barbosa, M.J., 2010. An outlook on microalgal biofuels. Science 329 (5993), 796–799. Yeesang, C., Cheirsilp, B., 2011. Effect of nitrogen, salt, and iron content in the growth medium and light intensity on lipid production by microalgae isolated from freshwater sources in Thailand. Bioresour. Technol. 102 (3), 3034–3040. Young, E.B., Beardall, J., 2005. Modulation of photosynthesis and inorganic carbon acquisition in a marine microalga by nitrogen, iron, and light availability. Can. J. Bot. 83 (7), 917–928. Zhila, N.O., Kalacheva, G.S., Volova, T.G., 2005. Influence of nitrogen deficiency on biochemical composition of the green alga Botryococcus. J. Appl. Phycol. 17 (4), 309–315.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Effect of phosphorus on biodiesel production from Scenedesmus obliquus under nitrogen-deficiency stress Fei-Fei Chu a, Pei-Na Chu b, Xiao-Fei Shen a, Paul K.S. Lam a,c, Raymond J. Zeng a,b,⇑ a
Advanced Laboratory for Environmental Research and Technology, USTC-CityU, Suzhou, PR China Department of Chemistry, University of Science and Technology of China, Hefei 230026, PR China c State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Nitrogen starvation enhanced
biodiesel productivity compared to nitrogen repletion. Biodiesel productivity was further strengthened by the supply level of phosphorus. More P was accumulated in cells under nitrogen starvation with sufficient P supply. A potential application to combine P removal with biodiesel production via microalgae.
a r t i c l e
i n f o
Article history: Received 20 August 2013 Received in revised form 29 October 2013 Accepted 7 November 2013 Available online 14 November 2013 Keywords: Scenedesmus obliquus FACHB-417 Nitrogen deficiency Phosphorus effect Biodiesel productivity Lipid trigger
a b s t r a c t In order to study the effect of phosphorus on biodiesel production from Scenedesmus obliquus especially under nitrogen deficiency conditions, six types of media with combinations of nitrogen repletion/depletion and phosphorus repletion/limitation/depletion were investigated in this study. It was found that nitrogen starvation compared to nitrogen repletion enhanced biodiesel productivity. Moreover, biodiesel productivity was further strengthened by varying the supply level of phosphorus from depletion, limitation, through to repletion. The maximum FAMEs productivity of 24.2 mg/L/day was obtained in nitrogen depletion with phosphorus repletion, which was two times higher than that in nutrient complete medium. More phosphorus was accumulated in cells under the nitrogen starvation with sufficient phosphorus condition, but no polyphosphate was formed. This study indicated that nitrogen starvation plus sufficient P supply might be the real ‘‘lipid trigger’’. Furthermore, results of the current study suggest a potential application for utilizing microalgae to combine phosphorus removal from wastewater with biodiesel production. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Renewable energy regarding biodiesel from microalgae has already been studied for decades (Wijffels and Barbosa, 2010). Various strategies have been used to increase algal biomass yield and/or lipid content in order to reduce the cost of biodiesel production. These strategies include nutrient limitation or starvation, ⇑ Corresponding author at: Department of Chemistry, University of Science and Technology of China, Hefei 230026, PR China. Tel./fax: +86 551 63600203. E-mail address: [email protected] (R.J. Zeng). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.11.013
high-intensity illumination, heterotrophic cultivation, etc. (Liu et al., 2008; Miao and Wu, 2006; Yeesang and Cheirsilp, 2011). Among these, nitrogen starvation is deemed as a major ‘‘lipid trigger’’ in green algae species (Sheehan et al., 1998). However, during subsequent decades, many high lipid-accumulating microalgal strains have been found to be associated with slow growth rate under nitrogen limited conditions, which leads to little improvement or even a decrease in overall oil productivity (El-Sheekh et al., 2013; Gordillo et al., 1998; Zhila et al., 2005). Some researchers believe that algal cultivation under sufficient nitrogen conditions is
242
F.-F. Chu et al. / Bioresource Technology 152 (2014) 241–246
more economical for lipid production than under deficient nitrogen conditions (Griffiths and Harrison, 2009; Mujtaba et al., 2012; Wijffels and Barbosa, 2010). Interestingly, our previous study found that phosphorus played an important role in enhancing lipid productivity of Chlorella vulgaris under nitrogen deficient conditions. With sufficient phosphorus supply, lipid productivity under nitrogen deficient conditions was 1.35 times higher than that of nutrient complete media. Moreover, phosphorus was luxuriously uptaken and accumulated as polyphosphate (Poly-P) (Chu et al., 2013). Actually, the wide distribution of Poly-P in many microorganisms, including blue–green algae and its accumulation under nutritional imbalance conditions, have been known for several decades (Harold, 1966). However, most researchers using stimulation method of nitrogen starvation for lipid production usually ignore the role of phosphorus (P), and this is one possible reason for the less than satisfactory lipid productivity under nitrogen depletion conditions (Rodolfi et al., 2009; Widjaja et al., 2009). It is unclear whether it is universally applicable for other microalgae that sufficient phosphorus supply over and above what is needed for growth is necessary in nitrogen deficiency media for higher lipid productivity. Meanwhile, phosphorus morphology in algal cells also needs to be investigated. Among microalgae, Scenedesmus obliquus contains oleic, palmitic and stearic acids as main fatty acids which are preferable for biodiesel production, and is also an excellent candidate for the economic combination of wastewater treatment and biodiesel production (Abd El Baky et al., 2012; Gouveia and Oliveira, 2009). Therefore, many researchers select S. obliquus as a potential and excellent source for lipid production. It has been found that the starvation or the limitation of nitrogen and/or phosphate could improve lipid content. However, large variations in lipid productivity exists amongst various studies perhaps because different experimental conditions and operational methods were applied (Abd El Baky et al., 2012; El-Sheekh et al., 2013; Ho et al., 2012; Mallick, 2009). For example, fatty acid content was enhanced by 54% over control under nitrogen deficiency, but its productivity was decreased due to the growth limitation (El-Sheekh et al., 2013). Li et al. (2010) reported that the lipid content of S. obliquus was the highest under nitrogen or phosphorus limitation, however, the highest lipid productivity was not achieved simultaneously. On the contrary, Breuer et al. (2012) found that S. obliquus showed the highest triacylglycerol (TAG) productivity among nine common strains under nitrogen starvation conditions, and the value was surprisingly 5.29 times that of nitrogen sufficient conditions. However, none of the researchers considered the effect of phosphorus under nitrogen starvation conditions. The aim of this study was to evaluate the role of P in biodiesel production from S. obliquus under nitrogen deficient conditions. Specifically, three levels of phosphate (sufficient, limited and deficient) were adopted under nitrogen sufficient and deficient conditions, respectively. The profiles of algal growth and nutrient absorption were monitored, and forms of phosphorus assimilated in cells were investigated. It was anticipated that P might play a role in enhancing the application of nitrogen deficiency or limitation for biodiesel production.
2. Methods 2.1. Strains and culture conditions S. obliquus FACHB-417 was obtained from Freshwater Algae Culture Collection of the Institute of Hydrobiology (FACHB), Chinese Academy of Sciences, Hubei province, China. The algae cultivation device was designed as described in a previous study (Chu et al., 2013). BG11 medium was autoclaved at 121 °C for 30 min and then
used to cultivate S. obliquus FACHB-417. The cultivation was maintained at 25 ± 2 °C under a light intensity of 120 lmol photons/ m2 s with photoperiodicity 16:8 (light for 16 h and dark for 8 h). Mixed gases (4% CO2 in air) were bubbled to the medium through a 0.20 lm PTFE membrane (SRP65, Sartorius, Germany) at a flow rate of 400 mL/min. The solution pH was kept at 6–8. 2.2. Experimental setup In order to investigate the effect of phosphorus on biomass and biodiesel production from S. obliquus, especially under nitrogen deficiency conditions, six types of media were adopted in this study, which included: sufficient nitrogen and sufficient phosphorus (N&P), sufficient nitrogen and phosphorus limited (N&P-lim), sufficient nitrogen and phosphorus deficiency (N&P-), nitrogen deficiency and sufficient phosphorus (N-&P), nitrogen deficiency and phosphorus limited (N-&P-lim), simultaneous nitrogen and phosphorus deficiency (N-&P-). S. obliquus FACHB-417 strain was pre-cultivated for 6 days and then centrifuged at 6000 rpm for 5 min. After washing with BG11 medium (without N and P) twice to remove residual orthophosphate and nitrate nitrogen, cells were suspended at an initial concentration of 160 mg/L in each of the six types of media. The concentration of phosphorus PO3 4 -P in the culture of N&P and N-&P experiments was 45 mg/L. Moreover, approximately 3.5 mg/L phosphorus was added on day 6 to induce a phosphorus limited condition for N&P-lim and N-&P-lim. The initial concentrations of nitrate and orthophosphate for these six media are shown in Table 1. The experiments for each type of medium lasted 16 days and operated in two duplicates. The liquid sample was collected with respect to time to determine biomass production, residual nitrate and orthophosphate concentrations. Moreover, algal cells were collected intermittently after centrifugation and then freeze-dried using a vacuum freeze dryer (HETO power Dry Pl3000, USA) to obtain algal powder. Subsequently, protein/carbohydrate/FAMEs contents and P content per 107 cells were measured. 2.3. Analytical methods 2.3.1. Determination of growth and nutrient consumption profiles Biomass sample of broth (10 mL) was filtered through a cellulose acetate membrane filter and then dried at 105 °C overnight. The dry weight of microalgae was obtained by the difference between the dry weight of the membrane blank and the loaded filter. Cell counting was conducted using an improved Neubauer haemocytometer slide under an inverted biological phase contrast microscope (Meiji TC5300, Japan). Algal powder (5 mg) was resuspended in distilled water and then digested using 5% potassium persulfate solution. Subsequently, the total phosphate contents were determined after all types of phosphorus were converted into ortho3 phosphate. The measurements of NO 3 -N; PO4 -P were conducted using a water quality autoanalyzer (Aquakem 200, ThermoFisher, Finland) according to standard methods (APHA, 1998). The assim3 ilation rates of NO 3 -N and PO4 -P were calculated using the linear regression slopes of the data with time. 2.3.2. Determination of starch content Freeze-dried algae powder (3 mg) was suspended in 2.5 mL of distilled water and autoclaved for 1 h at 135 °C for starch solubilization. Total starch in algal cells was analyzed using a commercial enzymatic Starch Assay Kit (SA-20, Sigma–Aldrich) based on amyloglucosidase digestion which converts starch to glucose. 2.3.3. Determination of crude protein content The crude protein content in algal cells was determined using the indirect method reported by Becker (1993). A correlation of
243
F.-F. Chu et al. / Bioresource Technology 152 (2014) 241–246 Table 1 The initial concentrations of nitrate and orthophosphate for six types of culture media. Initial concentration (mg/L) N NO 3 -N P PO3 4 -P a
N&P
N&P-lim
N&P-
N-&P
240
240
240
0
45
3.0a
0
45
N-&P-lim
N-&P-
0
0
3.0a
0
Approximately 3.5 mg/L phosphorus was added again on day 6 to induce a phosphorus limited condition for N&P-lim and N-&P-lim.
‘‘protein content = nitrogen content 6.25’’ was used to describe the relationship between protein content and nitrogen content. The nitrogen content of algal cells was measured using an elemental analyzer (Elementar Vario MACEO, Germany). 2.3.4. Determination of biodiesel production and 31P NMR Biodiesel was determined as fatty acid methyl esters (FAMEs) after direct transesterification adapted from Rodriguez-Ruiz et al. (1998). Lyophilized algal powder (20 mg) was collected and then freshly prepared transesterification reagent (a mixture of acetyl chloride and methanol (1:9 v/v), 2 mL), was added. Also internal standard methyl benzoate (0.5 g/L in hexane) was added after transesterification and the samples were analyzed using a gas chromatograph (Agilent 6890, CA). All the detailed steps and operating conditions are described in our previous study (Chu et al., 2013). The total FAMEs content, which is representative the oil content in the cells, was calculated as the percentage of dry biomass. FAMEs productivity was calculated based on Eq. (1):
FAMEs productivity ðmg=L=dayÞ Biomass production ðmgÞ FAMEs content ð%Þ ¼ Working volume ðLÞ Cultivation time ðdayÞ
ð1Þ
For the measurement of phosphorus forms in algal cells after 16-day cultivation, the samples were prepared using the method of perchloric acid extraction and then measured using 31P NMR. The details are given in our previous study (Chu et al., 2013). 2.4. Statistical analysis Statistical analysis was performed using Excel 2007 software (Microsoft, USA). Variables were reported as significant at 95% confidence (p-value less than or equal to 0.05). 3. Results and discussion 3.1. Algal growth profile Fig. 1 shows the growth profiles of S. obliquus FACHB-417 in six types of media. During the 16-day experiment, the growth rates of N&P, N&P-lim and N&P- were 104.0, 95.9 and 42.1 mg/L/day, respectively, while the rates for N-&P, N-&P-lim and N-&P- were 57.1, 52.4, 34.6 mg/L/day, respectively. To determine whether the effects were significant, p-values for the two variables (N supply and P concentration) were obtained after statistical analysis. Nitrogen supply (p = 3.3 106 < 0.05) and ortho-phosphate concentration (p = 8.63 106 < 0.05) were found to have significant effects on biomass growth. Significant interactions occurred between nitrogen supply and P concentration with p-value of 0.0005 (Fig. 1). The rates under nitrogen sufficient conditions were higher than the nitrogen starvation conditions with the same level of phosphorus supply. This phenomenon for S. obliquus was verified by other researchers as well. Mallick (2009) reported extremely little increase of biomass yield under conditions of N limitations or deficiencies, while El-Sheekh et al. (2013) found no growth of S. obliquus in the cultures devoid of nitrogen. Moreover, in both
Fig. 1. Profiles of biomass growth of S. obliquus FACHB-417 cultured in different types of media under the conditions of 4% CO2, 25 ± 2 °C and light intensity of 120 lmol photons/m2 s. Values shown are averages of two samples ± range.
nitrogen sufficient and starvation conditions, the rates of biomass increase under phosphorus sufficient conditions were slightly higher than under phosphorus limitation conditions, but were far greater than under phosphorus starvation conditions. These results indicated that phosphorus was vital for maintaining the normal metabolism of algal cells. 3.2. Nutrient assimilation The concentrations of nitrogen NO3 -N and phosphorus 3 PO4 -P in the media were determined and their assimilation profiles by S. obliquus are shown in Fig. 2. The concentration of NO 3 -N decreased rapidly from 240 mg/L to 144.7 mg/L in N&P and to 146.2 mg/L in N&P-lim, respectively, while it was almost unchanged in N&P- condition. Phosphorus concentration had a significant effect on nitrate assimilation (p = 0.001 < 0.05) (Fig. 2A). This phenomenon was also observed when C. vulgaris grew in phosphorus depletion media (Chu et al., 2013). It seems a very plausible hypothesis that the existence of phosphorus is essential for nitrate absorption by algal cells. Cellular energy transduction is closely associated with inorganic phosphate. Various aspects of metabolism including transportation of nitrate ion and proteins synthesis (nitrogen needed) would be down-regulated via adenylate limitation and plasmalemma H+-ATPase activity (Beardall et al., 2005). Moreover, the nitrate assimilation rate in N&P-lim almost equaled the rate in N&P (Table 2). It indicated that a small quantity of phosphorus was adequate to support the nitrogen assimilation. Fig. 2B illustrates the phosphorus assimilation capacity of S. obliquus FACHB-417 under different conditions. In N&P-lim and N-&P-lim, 3.5 mg/L phosphorus was added on day 6, and its concentration remained at a very low level again after two days of rapid consumption. As shown in Table 2, the overall phosphorus assimilation rate under N&P was 0.94 mg/L/day, which was even more than that in N-&P (0.78 mg/L/day), however, the nitrogen supply had no significant effect on the ortho-phosphate assimilation (p = 0.25 > 0.05) (Fig. 2B). This situation was quite different from C. vulgaris reported in our previous work (Chu et al., 2013).
244
F.-F. Chu et al. / Bioresource Technology 152 (2014) 241–246
Fig. 3. Phosphorus content per cell of S. obliquus FACHB-417 cultured with the media of N&P, N-&P, N&P-lim. Values shown are averages of two samples ± range.
Fig. 2. Profiles of nutrients assimilation by S. obliquus FACHB-417 cultured in different types of media under the conditions of 4% CO2, 25 ± 2 °C and light intensity 3 of 120 lmol photons/m2 s. (A) NO 3 -N. (B) PO4 -P. Values shown are averages of two samples ± range.
Table 2 Summary of the overall N and P consumption rates of six media during16-day cultivation. Rate (mg/L/day) N NO 3 -N P PO3 4 -P
N&P
N&P-lim
N&P-
N-&P
N-&P-lim
N-&P-
6.00
5.86
0.38
–
–
–
0.94
0.34
–
0.78
0.27
–
–, No data available.
A low phosphorus consumption rate of 0.49 mg/L/day was found in the control with complete nutrients while nitrogen starvation was associated with a strong phosphorus assimilation rate (1.87 mg/L/ day) in N-&P (Chu et al., 2013). 3.3. Phosphorus content in cell and
31
P NMR
S. obliquus FACHB-417 was investigated not only for its phosphorus absorption rate, but also for the phosphorus content and its form in cells. The phosphorus content inside the cells in N&P, N-&P and N&P-lim are shown in Fig. 3. After 16-day cultivation, phosphorus content gradually increased from 2.6 to 12.3 lg/ 107 cells under N-&P condition, stabilized at an average value of 5.4 lg/107 cells in N&P, but declined to 1.9 lg/107 cells in N&Plim. It demonstrated that nitrogen starvation provided a trigger for more phosphorus to be assimilated in S. obliquus cells in nitrogen starvation media, though phosphorus assimilation rates were quite similar between N&P and N-&P (Fig. 2B). The values of phosphorus content in these three conditions were significantly different (p = 0.0002 < 0.05) (Fig. 3). Li et al. (2010) also reported that the assimilation rate of TP was significantly enhanced as initial TN concentration decreased in the media.
Fig. S1 shows the 31P NMR spectra of S. obliquus under N&P and N-&P conditions. Inorganic orthophosphate (Pi) and organic phosphorus were observed distinctly in the two spectra. However, no Poly-P accumulation, which corresponded to the chemical shift mainly at 20.00 ppm, was found under both conditions. C. vulgaris showed Poly-P accumulation under nitrogen starved conditions (Chu et al., 2013). Inorganic polyphosphates have been found in organisms such as eubacteria, fungi, algae, protozoa and even in various tissues of higher plants and animals, and its accumulation was usually boosted under stress conditions including nitrogen depletion (Harold, 1966; Kulaev and Vagabov, 1983). Poly-P are thought to function not only as a phosphorus reserve, but also as an energy source reserve which enables cells to grow smoothly under stress conditions (Harold, 1966). However, inorganic polyphosphates are not obligatory cell components and may not be vitally important for organisms (Kulaev and Vagabov, 1983). It seems that Poly-P accumulation in microalgae is far from being universal. Meanwhile, more inorganic P was found in N-&P than in N&P, which was consistent with the trend of P contents in Fig. 3. 3.4. Composition of storage products Protein, starch and lipid are major storage products in microalgae. As shown in Fig. 4A, protein content reduced rapidly from 52.8% to an average value of 17.9% in the first 4 days under all nitrogen starvation conditions since nitrogen is a vital element for protein synthesis. Protein contents remained almost unchanged in N&P and N&P-lim but had a slight decline in N&P- which could be explained by the lack of metabolic energy attributed to phosphorus depletion. The starch level rose rapidly within 4 days and then slowly increased during the following 12 days, with final contents of 28.9%, 26.2% and 25.6% in N-&P, N-&P-lim and N-&P-, respectively. There was not much change (around 7%) under the conditions with sufficient nitrogen supply (Fig. 4B). In terms of FAMEs profile, accumulation lagged behind starch and increased dramatically after 4 days under all nitrogen depleted conditions, and the final contents of N-&P, N-&P-lim and N-&P- were 37.6%, 35.4% and 35.0%, respectively. A dramatic enhancement of lipid content from 10% to 43% had been observed by Mallick (2009) as well under N-deficient condition. Similar to starch, FAMEs content was almost unchanged under nitrogen sufficient conditions (Fig. 4C). Regarding the effect of P, it seemed to have a positive effect for starch and FAMEs as both had an estimated increase of 2% at the end of the cultivation process with sufficient P supply (N-&P) compared with limited P supply (N-&P-lim) or P depletion (N-&P-) (Fig. 4B and C). Statistical analysis indicated that nitrogen supply had a significant effect on protein, starch and FAMEs
F.-F. Chu et al. / Bioresource Technology 152 (2014) 241–246
245
Fig. 5. FAMEs productivity of S. obliquus FACHB-417 cultured with different types of media under the conditions of 4% CO2, 25 ± 2 °C and light intensity of 120 lmol photons/m2 s. Values shown are averages of two samples ± range.
Fig. 4. Storage products of S. obliquus FACHB-417 grown under different types of media under the conditions of 4% CO2, 25 ± 2 °C and light intensity of 120 lmol photons/m2 s. (A) Protein. (B) Starch and (C) total FAMEs. Values shown are averages of two samples ± range.
contents, while ortho-phosphate concentration had a significant effect on protein, but not on starch and FAMEs (Fig. 4). The lipid accumulation accompanied by starch increase was also observed in other algal species, such as S. obliquus, Chlorella zofingiensis, Phaeodactylum tricornutum and Neochloris oleoabundans, and all without exception showed that the accumulation of oil lagged behind that of starch (Breuer et al., 2012). This can be explained by an examination of biosynthesis metabolic pathways of starch and oil in Chlamydomonas reinhardtii that the biosynthesis of the two organic compounds competed not only for common carbon precursors, but also for metabolic energy and space in chloroplasts (Fan et al., 2012). 3.5. FAMEs productivity FAMEs productivity reflects the integration of biomass productivity and FAMEs content. As shown in Fig. 5, higher FAMEs
productivity was found under nitrogen starvation conditions than under nitrogen sufficient conditions with equal phosphorus content. Moreover, higher phosphorus supply resulted in higher FAMEs productivity under nitrogen deficiency (N-&P > N-&Plim > N-&P-). The highest FAMEs productivity was 24.2 mg/L/day in N-&P. In N-&P, FAMEs productivity was approximately two times greater compared to N&P during the whole cultivation period (Fig. 5). Nitrogen supply (p = 1.4 105 < 0.05) and orthophosphate concentration (p = 0.0003 < 0.05) were found to have significant effects on FAMEs productivity, however, no significant interactions occurred between these two variables (p = 0.25 > 0.05) (Fig. 5). Clearly, these results demonstrated that nitrogen starvation did enhance biodiesel productivity, which however contradicts most nitrogen deficient studies concerning S. obliquus (El-Sheekh et al., 2013; Li et al., 2010; Mallick, 2009). For example, El-Sheekh et al. (2013) reported that fatty acids productivity reduced sharply due to growth inhibition under nitrogen limitation despite the fact that a lipid content of 1.54 times greater was obtained. Phosphorus demonstrated a strong positive role in enhancing biodiesel productivity under both nitrogen depletion and sufficient conditions, and this observation was consistent with our previous study of C. vulgaris (Chu et al., 2013). Unfortunately, most studies in literature did not consider the importance of sufficient phosphorus supply for the enhancement of lipid productivity with nitrogen starvation stimulation. It is known that lipid synthesis in microalgae is via so-called CCMs (CO2 concentrating mechanisms), a pathway responsible for the acquisition and fixation of inorganic carbon from the environment, in which CO2 is assimilated and converted to organic compounds as lipid and carbohydrate. An enhancement of CCMs capacity in Dunaliella tertiolecta was observed under nitrogen stress, although the photosynthetic capacity was compromised (Young and Beardall, 2005). However, the CCMs activity in Chlorella emersonii was markedly down-regulated under phosphate limited conditions as it led to a slow growth rate and an increase of inorganic carbon (2005). Carbon acquisition is an ATP requiring process. It is likely that P limitation results in decreased ATP levels which reduce CCM activity, and finally decreases lipid productivity. 3.6. Implications of this work S. obliquus and C. vulgaris are two important and well studied green algae for lipid production. In our previous study on C. vulgaris (Chu et al., 2013), it was found that phosphorus uptake rates were 5.3 and 19.6 mg P/g biomass in N&P and N-&P, respectively, while
246
F.-F. Chu et al. / Bioresource Technology 152 (2014) 241–246
the rates for S. obliquus in this study were 9.1 and 13.0 mg P/g biomass, respectively. When comparing N-&P to N&P, lipid productivity was enhanced 1.35 times and 2 times for C. vulgaris and S. obliquus, respectively. Meanwhile, Poly-P was found in C. vulgaris, but not in S. obliquus. These findings indicated that the form of P inside the cells was not crucial for the enhancement of biodiesel production. In summary, both our studies on C. vulgaris and S. obliquus showed that P likely plays an important role in green algae in enhancing lipid productivity under nitrogen deficiency condition. Whether the role of P in the enhancement of biodiesel productivity can be extended to other types of algal species beyond green algae requires further investigation. Nevertheless, it indicates that nitrogen starvation plus sufficient P supply may be the real ‘‘lipid trigger’’ for microalgal biodiesel production. Furthermore, due to the faster P uptake rate under nitrogen starvation, it suggests a potential application for utilizing microalgae to combine phosphorus removal from wastewater with biodiesel production. For example, microalgae can grow normally in the first stage under nitrogen sufficient conditions, and then enter into the second stage with nitrogen deprivation and sufficient phosphorus to enhance biodiesel productivity and phosphorus removal. However, more investigations are required to verify this combined process, especially with regard to technical and economical aspects. 4. Conclusions This study demonstrated that nitrogen depletion was an effective way for stimulating biodiesel production from S. obliquus. Moreover, biodiesel productivity was further strengthened by altering the supply level of phosphorus from depletion, limitation, through to repletion. The highest FAMEs productivity of 24.2 mg/L/ day was obtained in N-&P, and the level was two times that of nutrient complete medium. Furthermore, more phosphorus was accumulated in cells under the condition of nitrogen starvation with sufficient phosphorus compared to nutrient complete medium, but no Poly-P formation was observed. This finding indicated that nitrogen starvation plus sufficient phosphorus supply might be the real ‘‘lipid trigger’’. Acknowledgements The authors would like to acknowledge the financial support of the Hundred-Talent Program of Chinese Academy of Sciences and the Program for Changjiang Scholars and Innovative Research Team in University. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2013. 11.013. References Abd El Baky, H.H., El-Baroty, G.S., Bouaid, A., Martinez, M., Aracil, J., 2012. Enhancement of lipid accumulation in Scenedesmus obliquus by optimizing CO2 and Fe3+ levels for biodiesel production. Bioresour. Technol. 119, 429–432.
APHA, 1998. Standard Methods for Examination of Water and Wastewater, twentieth ed. American Public Health Association, Washington, DC. Beardall, J., Roberts, S., Raven, J.A., 2005. Regulation of inorganic carbon acquisition by phosphorus limitation in the green alga Chlorella emersonii. Can. J. Bot.-Rev. Canadienne De Botanique 83 (7), 859–864. Becker, E.W., 1993. Microalgae: Biotechnology and Microbiology. Cambridge University Press. Breuer, G., Lamers, P.P., Martens, D.E., Draaisma, R.B., Wijffels, R.H., 2012. The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains. Bioresour. Technol. 124, 217–226. Chu, F.-F., Chu, P.-N., Cai, P.-J., Li, W.-W., Lam, P.K.S., Zeng, R.J., 2013. Phosphorus plays an important role in enhancing biodiesel productivity of Chlorella vulgaris under nitrogen deficiency. Bioresour. Technol. 134, 341–346. El-Sheekh, M., Abomohra, A.E.-F., Hanelt, D., 2013. Optimization of biomass and fatty acid productivity of Scenedesmus obliquus as a promising microalga for biodiesel production. World J. Microbiol. Biotechnol. 29 (5), 915–922. Fan, J., Yan, C., Andre, C., Shanklin, J., Schwender, J., Xu, C., 2012. Oil accumulation is controlled by carbon precursor supply for fatty acid synthesis in Chlamydomonas reinhardtii. Plant Cell Physiol. 53 (8), 1380–1390. Gordillo, F.J.L., Goutx, M., Figueroa, F.L., Niell, F.X., 1998. Effects of light intensity, CO2 and nitrogen supply on lipid class composition of Dunaliella viridis. J. Appl. Phycol. 10 (2), 135–144. Gouveia, L., Oliveira, A.C., 2009. Microalgae as a raw material for biofuels production. J. Ind. Microbiol. Biotechnol. 36 (2), 269–274. Griffiths, M.J., Harrison, S.T.L., 2009. Lipid productivity as a key characteristic for choosing algal species for biodiesel production. J. Appl. Phycol. 21 (5), 493–507. Harold, F.M., 1966. Inorganic polyphosphates in biology: structure, metabolism, and function. Bacteriol. Rev. 30 (4), 772. Ho, S.-H., Chen, C.-Y., Chang, J.-S., 2012. Effect of light intensity and nitrogen starvation on CO2 fixation and lipid/carbohydrate production of an indigenous microalga Scenedesmus obliquus CNW-N. Bioresour. Technol. 113, 244–252. Kulaev, I.S., Vagabov, V.M., 1983. Polyphosphate metabolism in microorganisms. Adv. Microb. Physiol. 24, 83–171. Li, X., Hu, H.Y., Gan, K., Sun, Y.X., 2010. Effects of different nitrogen and phosphorus concentrations on the growth, nutrient uptake, and lipid accumulation of a freshwater microalga Scenedesmus sp.. Bioresour. Technol. 101 (14), 5494–5500. Liu, Z.Y., Wang, G.C., Zhou, B.C., 2008. Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresour. Technol. 99 (11), 4717–4722. Mallick, S.M.N., 2009. Microalga Scenedesmus obliquus as a potential source for biodiesel production. Appl. Microbiol. Biotechnol. 84 (2), 281–291. Miao, X., Wu, Q., 2006. Biodiesel production from heterotrophic microalgal oil. Bioresour. Technol. 97 (6), 841–846. Mujtaba, G., Choi, W., Lee, C.-G., Lee, K., 2012. Lipid production by Chlorella vulgaris after a shift from nutrient-rich to nitrogen starvation conditions. Bioresour. Technol. 123, 279–283. Rodolfi, L., Zittelli, G.C., Niccolo‘, Bassi, Padovani, G., 2009. Microalgae for Oil: Strain Selection, Induction of Lipid Synthesis and Outdoor Mass Cultivation in a LowCost Photobioreactor. Biotechnology and Bioengineering 102 (1), 100–112. Rodriguez-Ruiz, J., Belarbi, E.H., Sanchez, J.L.G., Alonso, D.L., 1998. Rapid simultaneous lipid extraction and transesterification for fatty acid analyses. Biotechnol. Tech. 12 (9), 689–691. Sheehan, J., Dunahay, T., Benemann, J., Roessler, P., 1998. A Look Back at the US Department of Energy’s Aquatic Species Program: Biodiesel from Algae. National Renewable Energy Laboratory Golden. Widjaja, A., Chien, C.C., Ju, Y.H., 2009. Study of increasing lipid production from fresh water microalgae Chlorella vulgaris. J. Taiwan Inst. Chem. Eng. 40 (1), 13– 20. Wijffels, R.H., Barbosa, M.J., 2010. An outlook on microalgal biofuels. Science 329 (5993), 796–799. Yeesang, C., Cheirsilp, B., 2011. Effect of nitrogen, salt, and iron content in the growth medium and light intensity on lipid production by microalgae isolated from freshwater sources in Thailand. Bioresour. Technol. 102 (3), 3034–3040. Young, E.B., Beardall, J., 2005. Modulation of photosynthesis and inorganic carbon acquisition in a marine microalga by nitrogen, iron, and light availability. Can. J. Bot. 83 (7), 917–928. Zhila, N.O., Kalacheva, G.S., Volova, T.G., 2005. Influence of nitrogen deficiency on biochemical composition of the green alga Botryococcus. J. Appl. Phycol. 17 (4), 309–315.
