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Saline wastewater treatment by Chlorella vulgaris with simultaneous algal lipid accumulation triggered by nitrate deficiency
Accepted Manuscript Saline wastewater treatment by Chlorella vulgaris with simultaneous algal lipid accumulation triggered by nitrate deficiency Qiao-Hui Shen, Yu-Peng Gong, Wen-Zhe Fang, Zi-Cheng Bi, Li-Hua Cheng, Xin-Hua Xu, Huan-Lin Chen PII: DOI: Reference:
S0960-8524(15)00841-X http://dx.doi.org/10.1016/j.biortech.2015.06.050 BITE 15130
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
25 April 2015 9 June 2015 10 June 2015
Please cite this article as: Shen, Q-H., Gong, Y-P., Fang, W-Z., Bi, Z-C., Cheng, L-H., Xu, X-H., Chen, H-L., Saline wastewater treatment by Chlorella vulgaris with simultaneous algal lipid accumulation triggered by nitrate deficiency, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.06.050
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Saline wastewater treatment by Chlorella vulgaris with simultaneous algal lipid accumulation triggered by nitrate deficiency
Qiao-Hui Shen1, Yu-Peng Gong1, Wen-Zhe Fang 1, Zi-Cheng Bi1, Li-Hua Cheng1∗, Xin-Hua Xu1, Huan-Lin Chen2 1
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Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, P.R.China
Department of Chemical and Biochemical Engineering, Zhejiang University, Zhejiang 310027, PR China.
Revision Submitted to Bioresource Technology June, 2015
∗
Corresponding author: Tel.(fax): +86-571-88982025 E-mail address: [email protected] (L.-H. Cheng). 1
ABSTRACT
Chlorella vulgaris, a marine microalgae strain adaptable to 0-50 g L-1 of salinity, was selected for studying the coupling system of saline wastewater treatment and lipid accumulation. The effect of total nitrogen (TN) concentration was investigated on algal growth, nutrients removal as well as lipid accumulation. The removal efficiencies of TN and total phosphorus (TP) were found to be 92.2-96.6% and over 99%, respectively, after a batch cultivation of 20 days. To illustrate the response of lipid accumulation to nutrients removal, C. vulgaris was further cultivated in the recycling experiment of tidal saline water within the photobioreactor. The lipid accumulation was triggered upon the almost depletion of nitrate (<5 mg L-1), till the final highest lipid content of 40%. The nitrogen conversion in the sequence of nitrate, nitrite, and then to ammonium in the effluents was finally integrated with previous discussions on metabolic pathways of algal cell under nitrogen deficiency.
Keywords: Nitrogen deficiency; Lipid accumulation; Saline wastewater; Microalgae; Chlorella vulgaris
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1. Introduction Saline wastewater，rich in both salt ( mainly NaCl) and nutrients，is often generated during the manufacture of food-processing, leather and petroleum industry, professionally known as produced waters (Lefebvre and Moletta, 2006). Utilization of seawater as part of water supply in situations where high quality water is unnecessary also promotes to generate large amounts of saline wastewaters. High salinity has been proved to strongly inhibit the metabolism of nitrifying bacteria and cause a failure to nutrients removal from saline wastewaters (Uygur and Kargi, 2004). In many studies, halophilic organisms were considered adaptable to treat saline wastewater in order to prevent pollutants (Duan et al., 2015; Zhang et al., 2014). Compared with screening of the organisms acclimated to saline wastewater, utilization of marine microalgae would be more manipulable and economical. Marine microalgae are usually efficient in removing nitrogen and phosphorus from wastewater, and have the ability to avoid contamination by biological pollutants such as bacteria, virus and zooplankton. Biological pollutants contamination has become a big constraint in massive microalgal cultivation, especially when cultured in wastewater (Wang et al., 2013; Yu et al., 2014). In our previous research, Scenedesmus obliquus was also found difficult to cultivate in the real municipal wastewater mainly due to the inevitable contamination of bacteria (Shen et al., 2015). To control infection and contamination of the biological pollutants, high salinity had been considered as one of solutions (Wang et al., 2013). Therefore, marine microalgae have the potential to play an important role in
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saline municipal wastewater treatment. Meanwhile, microalgae can achieve potentially high yield of lipid and high economic value of harvested biomass if further produced for fertilizers, biogas, and so on (Cai et al., 2013). The coupling system of saline wastewater treatment and lipid production based on microalgae can be a promising solution to the water as well as energy crises (Li et al., 2010c). However, in the coupling system, how to ameliorate the performance of both nutrients removal and lipid accumulation is a central concern. To achieve high efficiency of nitrogen and phosphorus removal and to obtain high lipid productivity are the two purposes of the coupling system. To increase nutrients removal efficiency and lipid productivity simultaneously, Chlorella vulgaris, a strain of highly productive oleaginous marine microalgae is selected for studying the coupling system of lipid accumulation and saline wastewater treatment. Green unicellular microalgae have long been proposed as a potential renewable fuel source to produce biodiesel (Benemann et al., 1977). Chlorella vulgaris, when grown under nitrogen-sufficient conditions, produced biomass of approximately 0.17-0.18 g L-1 d-1, on a dry cell basis. Under nitrogen limitation, Chlorella vulgaris yielded total lipid content of 22%-63% by weight, mainly TAGs that are suitable for further production of biodiesel (Rodolfi et al., 2009; Stephenson et al., 2010). Meanwhile, Chlorella has been reported to be tolerant to various sewage effluent conditions and efficient in removing nutrients, such as nitrogen and phosphorus, from wastewater (Tuantet et al., 2014; Valderrama et al., 2002). Noting that Chlorella
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vulgaris has not been utilized for nitrogen and phosphorus removal in saline wastewater. To induce the lipid accumulation in Chlorella vulgaris, the regulation strategy by nutrient limitations or deficiencies has been widely studied, including nitrogen, phosphate and sulfate, among which nitrogen deficiency is regarded as the most efficient approach. Li et al. (2010a) observed lipid accumulation as high as 30% of algal biomass for nitrogen limitation (initial TN of 2.5 mg L-1) by Scenedesmus sp. LX1. Stephenson et al. (2010) reported that leaving the algal cells in a medium containing 200 mg L-1 nitrate for 10 to 20 days would cause the natural depletion of nitrogen and thus enhance the lipid content, reaching up to 46% of TAG (triacylglycerol) in dry cell mass. However, upon what low level of nitrogen concentration the lipid would be triggered has not been reported in literature to our best knowledge. In this study, the effect of initial salt concentration on cell growth and lipid accumulation was first studied to investigate the potential of C. vulgaris for saline wastewater treatment. The nutrients removal and lipid accumulation, along with the nitrogen deficiency, were simultaneously investigated with different initial total nitrogen (TN) of 10 to 40 mg L-1. The recycling experiment within the photobioreactor was also investigated for a better understanding of lipid accumulation mechanisms. A real saline wastewater, the tidal saline water from Qiantang River (Zhejiang Province, China), was finally chosen to verify the feasibility of saline municipal wastewater treatment using C. vulgaris.
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2. Materials and methods 2.1 Algal species and municipal saline wastewater The marine microalgae Chlorella vulgaris, provided by the Key Laboratory of Marine Biotechnology, Ningbo University (Zhejiang, China), were cultivated. Before experiment, the strain was preserved in the artificial seawater medium (SM medium): 30 g L-1 instant sea salt, 2.5 mg L-1 FeSO47H2O, 1000 mg L-1 KNO3, 0.25 mg L-1 MnSO4, 10 mg L-1 KH2PO4, 6×10-3 mg L-1 Vitamin B1, 10 mg L-1 Na2EDTA, 5×10 -5 mg L-1 Vitamin B12. Wastewater environment was first provided by simulated municipal saline wastewater. NO3--N was used as the nitrogen source. In addition to nitrogen, the adapted artificial wastewater was prepared by dissolving the following chemicals: 13 mg L-1 KH2PO4 (3 mg L-1 PO43--P), 37.5 mg L-1 MgSO4·7H2O, 18 mg L-1 CaCl2·2H2O, 3 mg L-1 citric acid, 5 mg L-1 FeSO4·7H2O, 0.5 mg L-1 EDTA, 10 mg L-1 Na2CO3, 30 g L-1 sea salt and 1.0 mL L-1 A5+Co solution. The A5+Co solution contained 2.86 g L-1 H3BO3, 1.81 g L-1 MnCl2·H2O, 222 mg L-1 ZnSO4·7H2O, 79 mg L-1 CuSO4·5H2O, 390 mg L-1 Na2MoO4·2H2O, and 49 mg L-1 Co(NO3)2·6H2O. The initial pH was adjusted to 7.0-8.0. The tidal saline wastewater samples from Qiantang River were collected 1 hour before the tide coming, 1 hour and 2 hours after the tide during the tide period, with the average water conductivity of 2645 µS cm-1, 6217 µS cm-1 and 8810 µS cm-1, respectively. The TN concentrations ranged between 5.0 mg L-1 and 7.0 mg L-1, while
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the TP concentrations were about 0.30-0.35 mg L-1. 2.2 Experiment design The microalgae were first cultivated in a batch mode using artificial wastewater as nutrient supply. The experiments were conducted in 500 mL flasks containing 250 mL autoclaved artificial wastewater. The algal inoculums of 100 mL were centrifuged at 11180 × g for 10 min at 4°C, and the supernatant was discharged. The pellet of algal cells was then re-suspended into the artificial wastewater in a 500 mL flask. Nine flasks with an initial NaCl of 0-90 mg L-1 were prepared for the experiment to clarify the effect of initial NaCl concentration on cell growth and lipid content, while the initial TN and TP was 20 mg L-1 and 3 mg L-1, respectively. Four flasks with an initial TN of 10 mg L-1, 20 mg L-1, 30 mg L-1 and 40 mg L-1 were then prepared to study the effect of initial TN on algal growth, lipid accumulation and the removal of nitrogen and phosphorus. The initial TP and NaCl concentration for all the samples was kept at 3 mg L-1 and 30 g L-1. The second period was started with the same initial concentrations as the first one. The flasks of the two experiments mentioned above were then shaken under the continuous illumination of 40 µmol photons m-2 s-1 at 25°C. The experiments were conducted three times to verify the data reliability. In the recycling cultivation experiment, the culture system used was a cylindrical plexiglass photobioreactor with a working volume of 1 L (380 mm in height, 60 mm in diameter). Six fluorescent lamps were arranged around the photobioreactor to supply illumination. At the bottom of the reactor, a gas sparger was fixed and room air was
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provided by a compressor. The experiment was conducted in three periods. After each period, the microalgae were retained in the reactor and the supernatant was discharged. Then the new wastewater with the initial nitrogen concentration of 20 mg L-1 was added into the reactor to begin a new period.
2.3 Analytical methods The biomass was evaluated by dry weight DW (g L-1), and its relationship with the optical density (OD) at 450 nm was referred as Eq. (1) (Fan et al., 2008): (1)
DW = 0.49 × OD450
The specific growth rate (µ，d-1) was used to describe the algal growth (Eq. (2)) (Jiang et al., 2011):
µ=
ln( DWt − DW0 ) t − t0
(2)
where DWt and DW0 were the dry weight at the end and the beginning of the exponential phase, respectively, and t-t0 was the duration time of the exponential phase. The lipid concentration L (g L-1) was measured by a high throughput Nile red method (Feng et al., 2013). The microalgae stained by Nile red (9-diethylamino-5H-benzo[a]phenoxazine-5-one) were put into water bath at 37°C for 10 minutes, and then determined by spectrofluorophotometer (Lengguang Tech. F96Pro). The relationship between fluorescence intensity F at 585 nm and L (g L-1) was determined using triolein as the lipid standard, as shown in Eq. (3):
F = 6 ×104 L + 25.56
(3) 8
The lipid content (% dry weight) was calculated from dividing L (g L-1) by dry weight DW (g L-1), as shown in Eq. (4):
Lipid content =
L × 100% DW
(4)
By dividing the difference in lipid (∆L) by the time interval (∆t, days), the lipid productivity (mg L-1 d-1) was obtained, as shown in Eq. (5).
Lipid productivity =
∆L ×1000 ∆t
(5)
For the nutrient removal analysis, the samples from flasks were centrifuged at 11180 × g for 10 min to determine TN, NO3-–N, NO2-–N, NH4+–N and TP in the supernatants. All the measurements were conducted according to Chinese State Standard Testing Methods (State Environmental Protection Administration, 2002).
3. Results and discussion To verify the feasibility of saline municipal wastewater treatment using Chlorella vulgaris, the effect of initial salt concentration on cell growth and lipid accumulation was first investigated. The effect of initial TN on the algal growth, nutrients removal and lipid accumulation was studied to illustrate the lipid accumulation in response to nitrogen stress.
3.1 Effect of salinity on algal growth and lipid accumulation Since the salt concentration of natural habitat of C. vulgaris is 34 g L-1, its growth
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and lipid accumulation might be affected by salinity. The present study focused on the adaptation of C. vulgaris to variation of saline concentrations and their effect on the growth and lipid production. The flask cultivations were performed with several initial salt concentrations. As shown in Fig. 1, the dry weights of C. vulgaris remained stable at around 1.5 g L-1 in the salt concentration range of 0-50 g L-1. There was no significant effect of salinity on the algal growth between 0-50 g L-1. The dry weights under the conditions of 65 and 80 g L-1 were 0.9511 and 0.7908 g L-1, respectively. An apparent growth inhibition was observed when the salt concentration was higher than 50 g L-1. Consequently, the salt concentration lower than 50 g L-1 was considered appropriate for C. vulgaris to achieve high dry weight of biomass. The lipid content of biomass harvested at the end of cultivation under each condition was also shown in Fig.1. The lipid content of microalgae cultured without NaCl additional was only about 8%. The bar chart showed that there was a gradual increase in lipid content with the increase of salt concentration (below 50 g L-1). It indicated that the C. vulgaris was adaptable to 0-50 g L-1 of salinity with an increased production of lipid and might be suitable to treat saline wastewater with such a range of salinity.
3.2 Effect of initial TN on algal growth, lipid accumulation and the removal of nitrogen and phosphorus Fig. 2 reveals the algae growth, lipid accumulation and the removal of nitrogen and
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phosphorus under different initial nitrogen concentrations in batch culture. The experiment was conducted three times to verify the data reliability. The second period was started with the same initial concentrations as the first one. As shown in Fig. 2(a), C. vulgaris grew faster when more nitrogen was initially supplied. After each period of cultivation, the algal dry weight reached about 1.8 g L-1 at the initial TN of 40 mg L-1. The TN and TP concentrations in the culture medium decreased rapidly with the growth of cells as shown in Fig. 2 (c) and (d). The concentration of TP decreased more rapidly than that of TN, regardless of the initial TN concentrations. Similar sudden decrease of TP at the beginning of experiment has been reported by Li et al. (2010a) that TP decreased from 1.3 mg L-1 to 0.8 mg L-1 during the first day of cultivation. The phosphorus removal from the synthetic wastewater was not due to precipitation as chelating chemicals were added to prevent precipitation occurring at high pH levels. This meant that the phosphorus had been removed due to a combination of growth, adsorption and luxury uptake of phosphorus (Powell et al., 2009). The removal efficiencies of TN and TP at the end of each batch were calculated to be 92.2~96.6% and nearly 100%, respectively, when the initial concentrations were controlled at TN of 10~40 mg L-1 and TP of 3 mg L-1, as shown in Fig. 2 (c) and (d), demonstrating its efficiency in removing nitrogen and phosphorus from saline wastewater. In the first period, after a batch cultivation of approximately 2, 7, 10 days, the TN was lower than 15 mg L-1 for the initial TN of 20, 30, 40 mg L-1, respectively. In
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the second period, the TN was lower than 15 mg L-1 after a cultivation of approximately 1, 2, 3 days for the initial TN of 20, 30, 40 mg L-1, respectively. The TN removal efficiency in the second batch was apparently higher compared with that in the first batch which could be explained by the starvation period of about 10 days in the first batch. Fig. 2 showed that in the first period the highest removal rate of TN was 3.46 mg L-1 d-1 at the initial TN of 20 mg L-1, and the highest removal rate of TP was 2.26 mg L-1 d-1 at the initial TN of 40 mg L-1, indicating the high efficiency of the coupling system by Chlorella vulgaris in terms of nutrients removal. Although high removal efficiencies of nutrients were reported both with nitrate and ammonium as the nitrogen source, the removal rate of nutrients could be sensitive to the nitrogen source. Li et al. (2010b) reported a lower removal rate of TN and TP with ammonium as the nitrogen source, compared with nitrate as the nitrogen source. NH4+-N utilization produced H+ to cause acid pH, which would inhibit the algal growth during stable phase, thus reducing the removal rate of nutrients. The algal lipid content increased as shown in Fig. 2 (b) while the nitrogen was gradually removed as shown in Fig. 2 (c). By comparison of Fig.2 (b) and (c), the lipid accumulation was clearly observed at the almost depletion of the nitrogen. For example, in the first batch at the initial TN of 10 mg L-1, the nitrogen was almost depleted 2-4 days after inoculation. Induced by nitrogen stress, the lipid began to accumulate remarkably from the fifth day. Similar phenomena were observed at the initial TN of 20, 30, 40 mg L-1. After 20 days of batch cultivation in the first period, the final lipid
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contents were measured to a range from 9.76% for initial TN of 40 mg L-1 to 33.07% for initial TN of 10 mg L-1, showing the lower the initial TN, the earlier nitrogen deficiency and the higher final lipid content. Compared with the first batch at the initial TN of 10 mg L-1, the nitrogen was consumed faster and depleted within one day in the second batch because of the 10 days starvation period in the first batch. Meanwhile, the lipid decreased in the first day and began to accumulate from the second day simultaneously. With a daily culture replacement of 1.0 L in a column aeration photobioreactor, Feng et al. (2011) reported C. vulgaris culture achieved the highest lipid content of 42%. Using Scenedesmus sp. cultivated at the initial TN of 2.5 mg L-1, Li et al. (2010a) obtained the lipid content of about 30%. The two batches of experiment and frequent monitoring of lipid revealed that high lipid content resulted from a lipid accumulation in response to TN concentration. In this work, the lipid was found to accumulate when the nitrogen concentration was lower than about 5 mg L-1 and the lipid content would decrease correspondingly with the nitrogen concentration exceeding 5 mg L-1, clearly showing that lipid was boosted at the critical nitrogen deficiency point. Although many studies had discussed the effect of nitrate limitation on fatty acid production by microalgae, our research focused on the the specific concentration of nitrate in response to the lipid accumulation by the cycling experiments, which has been rarely reported to the best of our knowledge. The experiment also demonstrated that cultivating the microalgae in the saline municipal wastewater using batch mode could not only further reduce the nutrients including
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nitrogen and phosphorous, but also trigger the lipid accumulation without the further-assisted inducing methods, such as nitrogen-free environment (Stephenson et al., 2010) or strong irradiance (Hu et al., 2008). Stephenson et al. (2010) reported that rather than abruptly transfer the cells into a cultural medium lacking nitrogen, leaving the cells in a medium containing 200 mg L-1 nitrate for 10 to 20 days would enhance the lipid content (reached up to 46% of TAG in dry cell mass). The lipid content was found to accumulate more efficiently when the algal cells consumed the nitrogen in the medium naturally, which could be widely used in batch mode for boosting lipid content in biomass along with wastewater treatment.
3.3 Recycling microalgae in the photobioreactor As the shaking of flasks could not meet the need of C. vulgaris on carbon source, for example CO2, the cultivation of microalgae was further conducted in the photobioreactor with air aeration. The capabilities of algae growth, lipid accumulation and nitrogen removal by C. vulgaris were further attested in the photobioreactor. The recycling experiment within the photobioreactor was also investigated for a better understanding of lipid accumulation mechanisms. The algal biomass productivity, lipid productivity, TN and TP removal rates of each batch were investigated (Table 1 and Fig. 3). As shown in Fig. 3, the microalgae kept growing within all of the three periods. As shown in Table 1, the dry weight increased during operation, from 0.5929 g L-1 at the beginning to 1.038 g L-1 at the end. The
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specific growth rate of the second batch was 1.51 d-1, which was much higher than that of the other two batches. As shown in Fig. 3, the microalgae entered the decline phase and the dry weight began to decrease after 5 days in the third batch. Since biomass increased continuously in the system during the treatment process, the removal efficiencies of nitrogen and phosphorus for the three batches increased at the same time. The removal rates of TN and TP in the third batch were 8.63 mg L-1 d-1 and 2.27 mg L-1 d-1, respectively, which were both the highest during the three batches. The nitrogen in the newly added wastewaters was instantly removed in less than 3 days at the beginning of each period. The lipid content varied against the nitrogen concentration. In response to the high nitrogen concentration in the newly added wastewater, the lipid content dropped a little. As soon as the nitrogen was almost depleted, the lipid content was boosted dramatically to a higher level. After the three periods, the lipid content achieved 40%, which was higher than that of the microalgae cultivated in the flasks. The maximum lipid productivity of the three periods were 20.15 mg L-1 d-1, 32.33 mg L-1 d-1 and 54.25 mg L-1 d-1, respectively. The highest lipid productivity in the third batch was due to the high nitrogen removal efficiency and the large quantity of biomass.
3.4 Nutrients removal from tidal saline water The tidal saline water from Qiantang River was further chosen to verify the feasibility of saline municipal wastewater treatment using C. vulgaris. Qiantang River
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flows into the East China Sea via Hangzhou Bay and is well known for the large tidal bore. Water quality of Qiantang River is affected seriously by seawater intrusion into Qiantang River during tidal periods and the industrial wastewater and sewage excessive discharge in the upstream and downstream of Hangzhou City. Fig. 4 exhibited the variations in algal growth (a), lipid accumulation (b), nitrogen (c) and phosphorus (d) removal over three batch runs when cultivated within saline water. Fig. 4(a) and (b) exhibited the algal dry weight and lipid content over the three batches. The biomass dry weight increased gradually and reached about 0.34 g L-1 at the end of Batch 3, with slight difference between different runs of saline wastewater. The lipid content increased with increasing salinity, and reached 35% when cultured with saline water of 8810 µS cm-1. The results in Fig. 4 (c) and (d) demonstrated that C. vulgaris was able to remove total nitrogen and total phosphorus efficiently from saline water. The removal efficiency of TP was approximately 100% during the entire experiment which was not surprising as higher concentrations of TP were removed completely from artificial saline wastewater treatment experiments. The enhancement of lipid accumulation was not observed when cultured with water before the tide coming (2645 µS cm-1), even though TN had been removed below 5 mg L-1 (Fig. 4(b) and (c)). It indicated that an appropriate amount of salinity was essential for lipid accumulation of C. vulgaris under nitrogen deficiency, as already shown in Fig. 1. Our previous study proved it difficult to cultivate Scenedesmus obliquus in the real secondary municipal wastewater and the cells even disrupted after 7 days due to the contamination of bacteria (Shen et al., 2015). However, the marine C. vulgaris could remove nutrients and accumulate lipid efficiently within real tidal saline for three batches and remained active after 10 days in this study, in spite of the low 16
concentrations of nutrients within real tidal saline water. The difference showed the advantage of C. vulgaris to avoid contamination by bacteria and high salinity helped to control contamination by biological pollutants.
3.5 The potential mechanism of lipid accumulation triggered by nitrogen deficiency Although the lipid accumulation was clearly boosted at the point of nitrogen deficiency as shown in the Fig. 2 and Fig. 3, it was necessary to determine whether the deficiency of nitrate, ammonium, or other forms of nitrogen triggered lipid. The changes in NO3-–N, NO2-–N, NH4+–N for the initial TN of 20 mg L-1 were shown in Fig. 5. The NO3-–N was finally converted to NH4+–N in order to be utilized by C. vulgaris, during which the NO2-–N was produced. After the sixth day, the nitrate was almost depleted, while NH4 +–N remained at a relatively stable level. An increase of lipid was then observed after the sixth day. Therefore, although the nitrate could be converted to other forms of nitrogen after algal inoculation, the lipid accumulation was a response to the specific depletion of nitrate. Based on those from literature (Hu et al., 2008; Kliphuis et al., 2012; Li et al., 2012; Msanne et al., 2012), the potential mechanism diagram of lipid accumulation triggered by nitrogen stress was summarized as shown in Fig. 6. Ambient CO2, sun light and external nitrate/ammonium are normally utilized by microalgae as carbon, energy and nitrogen source, respectively, for maintaining life cycles including those involved in calvin cycle in chloroplast, tricarboxylic acid cycle (TCA cycle) and pentose phosphate pathway (PPP). A conversion of nitrogen from NO3-–N, to NO2-–N, and then to NH4+–N 17
had been observed in this work before they were transported into cytomembrane as shown in Fig. 6. The phenomenon was consistent with the result that microalgae prefer ammonium nitrogen to nitrate (Li et al., 2010b). When external nitrogen source was deficient for microalgal growth, the activity of calvin cycle involved in photosynthesis and electron transport was restrained as shown in blue lines while both the PPP and TCA cycles were strengthened as shown pink lines, resulting in low photosynthesis rate and a conversion from other components into lipid (Li et al., 2012; Msanne et al., 2012). Notice that Li et al. (2012) also found that carbohydrate was regarded as main carbon source for lipid synthesis as shown by the vertical arrow in Fig. 6, based on a decrease of intracellular carbohydrate content and the difference of relative genes’ transcription level between nitrogen-rich and nitrogen-free. This shift of metabolism to neutral lipids storage was an immediate response of microalgae to the stress from the cultural environment and served as a reserve for rebuilding the cell after the stress (Rodolfi et al., 2009; Roessler, 1990). Although the conversion of carbohydrate, protein and Chlorophyll through TCA cycle for further lipid synthesis has been reported, more information is still required for the metabolic pathways and functional genes involved in this process.
4. Conclusions The marine microalgae strain C. vulgaris was successfully used to treat saline wastewater including the tidal saline water. The lipid accumulation was triggered at the
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point of nitrate concentration below 5 mg L-1. The lipid content increased up to 40% and the maximum lipid productivity was 54.0 mg L-1 d-1 at the end of the recycling system in the photobioreactor. This study also showed the feasibility of C. vulgaris to remove TN and TP efficiently from real saline wastewater owing to its ability to avoid contamination of bacteria.
Acknowledgement This work is based upon research supported by Important National Science & Technology Specific Projects (2014ZX07101-012-04), the National Natural Science Foundation of China (No. 21106130, No. 21276221) and the Doctoral Discipline Foundation for Young Teachers in the Higher Education Institutions of Ministry of Education (201101011120074). We gratefully acknowledge to the Key Laboratory of Marine Biotechnology, Ningbo University (Zhejiang, China) for generously presenting Chlorella vulgaris.
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Biotechnol. 27(1), 59-63. Li, Y.J., Fei, X.W., Deng, X.D., 2012. Novel molecular insights into nitrogen starvation-induced triacylglycerols accumulation revealed by differential gene expression analysis in green algae Micractinium pusillum. Biomass Bioenerg. 42, 199-211. Msanne, J., Xu, D., Konda, A.R., Casas-Mollano, J.A., Awada, T., Cahoon, E.B., Cerutti, H., 2012. Metabolic and gene expression changes triggered by nitrogen deprivation in the photoautotrophically grown microalgae Chlamydomonas reinhardtii and Coccomyxa sp. C-169. Phytochemistry 75, 50-59. Powell, N., Shilton, A., Chisti, Y., Pratt, S., 2009. Towards a luxury uptake process via microalgae Defining the polyphosphate dynamics. Water Res. 43(17), 4207-4213. Rodolfi, L., Zittelli, G.C., Bassi, N., Padovani, G., Biondi, N., Bonini, G., Tredici, M.R., 2009. Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol. Bioeng. 102(1), 100-112. Roessler, P.G., 1990. Environmental control of glycerolipid metabolism in microalgae: commercial implications and future research directions. J. Phycol. 26(3), 393-399. Shen, Q.H., Jiang, J.W., Chen, L.P., Cheng, L.H., Xu, X.H., Chen, H.L., 2015. Effect of carbon source on biomass growth and nutrients removal of Scenedesmus obliquus for wastewater advanced treatment and lipid production. Bioresour. Technol. doi:10.1016/j.biortech.2015.04.053. State Environmental Protection Administration, 2002. Monitoring Method of Water and Wastewater, 4th edition. China Environmental Science Press, Beijing, P.R.China, pp.243-248, 254-284. Stephenson, A.L., Dennis, J.S., Howe, C.J., Scott, S.A., Smith, A.G., 2010. Influence of nitrogen-limitation regime on the production by Chlorella vulgaris of lipids for biodiesel feedstocks. Biofuels 1(1), 47-58. Tuantet, K., Temmink, H., Zeeman, G., Janssen, M., Wijffels, R.H., Buisman, C.J.N., 2014. Nutrient removal and microalgal biomass production on urine in a short light-path photobioreactor. Water Res. 55, 162-174. 21
Uygur, A., Kargi, F., 2004. Salt inhibition on biological nutrient removal from saline wastewater in a sequencing batch reactor. Enzyme Microb. Technol. 34 (3-4), 313-318. Valderrama, L.T., Del Campo, C.M., Rodriguez, C.M., de- Bashan, L.E., Bashan, Y., 2002. Treatment of recalcitrant wastewater from ethanol and citric acid production using the microalga Chlorella vulgaris and the macrophyte Lemna minuscula. Water Res. 36(17), 4185-4192. Wang, H., Zhang, W., Chen, L., Wang, J., Liu, T., 2013. The contamination and control of biological pollutants in mass cultivation of microalgae. Bioresour. Technol. 128(1), 745-750. Yu, Y., Wu, Y.H., Zhu, S.F., Hu, H.Y., 2015. The bioavailability of the soluble algal products of different microalgal strains and its influence on microalgal growth in unsterilized domestic secondary effluent. Bioresour. Technol. 180, 352-355. Zhang, X.H., Gao, J., Zhao, F.B., Zhao, Y.Y., Li, Z.S., 2014. Characterization of a salt-tolerant bacterium Bacillus sp. from a membrane bioreactor for saline wastewater treatment. J. Environ. Sci. 26(6), 1369-1374.
22
Table captions
Table 1. Algal biomass production, lipid accumulation and nutrients removal of the three batches cultivation with artificial wastewater in photobioreactors
23
Figure captions Fig. 1 Effect of initial salinity on the algal growth and the lipid accumulation. (initial nitrate concentration: 20 mg L-1, initial phosphorus concentration: 3 mg L-1, light intensity: 40 µmol photons m-2 s-1, temperature: 25 °C) Fig. 2 Effect of initial TN concentration on the algal growth within two batches (a), the lipid accumulation (b), the nitrogen (c) and the phosphorus (d) removal. (initial NaCl concentration: 30 g L-1, initial phosphorus concentration: 3 mg L-1, light intensity: 40 µmol photons m-2 s-1, temperature: 25 °C) Fig. 3 Changes in algal growth, nitrogen removal and lipid accumulation in biomass over three batch runs in the photobioreactor. (initial nitrogen concentration: 20 mg L-1, initial phosphorus concentration: 3 mg L-1, light intensity: 40 µmol photons m-2 s-1, temperature: 25 °C) Fig. 4 Changes in algal growth (a), lipid accumulation (b), nitrogen (c) and phosphorus (d) removal over three batch runs when cultivated with tidal saline water. Fig. 5 Time course of NO3--N, NO2--N, NH4+-N concentrations for the initial TN of 20 mg L-1. Fig. 6 Schematic of lipid accumulation triggered by nitrogen deficiency. (TCA cycle, PPP, GAP and G6P refer to tricarboxylic acid cycle, pentose phosphate pathway, glyceraldehyde 3-phosphate and glucose 6-phosphate, respectively.)
24
Table 1 Algal biomass production, lipid accumulation and nutrients removal of the three batches cultivation with artificial wastewater in photobioreactors Photobioreactor Stage 1
Stage 2
Stage 3
Max. biomass concentration (g L-1) 0.5929
0.9070
1.0380
Specific growth rate (d-1)
1.09
1.51
1.35
Lipid content (%)
24.1
31.4
40.1
Lipid productivity (mg L-1 d-1)
20.15
32.33
54.25
Removal rate of TP (mg L-1 d-1)
1.32
2.09
2.27
Removal rate of TN (mg L-1 d-1)
7.61
17.29
8.63
25
Highlights:
1. C. vulgaris was found suitable for saline wastewater treatment.
2. Salinity was essential for lipid accumulation of C. vulgaris.
3. Lipid content was boosted at point of nitrate deficiency by Nile red method.
4. High nutrient removal efficiencies could be obtained by recycling cultivation mode.
5. Pollutants in a real tidal saline water were efficiently utilized by microalgae.
26
27
a
b
c
d
28
29
a
b
c
d
30
31
32
S0960-8524(15)00841-X http://dx.doi.org/10.1016/j.biortech.2015.06.050 BITE 15130
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
25 April 2015 9 June 2015 10 June 2015
Please cite this article as: Shen, Q-H., Gong, Y-P., Fang, W-Z., Bi, Z-C., Cheng, L-H., Xu, X-H., Chen, H-L., Saline wastewater treatment by Chlorella vulgaris with simultaneous algal lipid accumulation triggered by nitrate deficiency, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.2015.06.050
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Saline wastewater treatment by Chlorella vulgaris with simultaneous algal lipid accumulation triggered by nitrate deficiency
Qiao-Hui Shen1, Yu-Peng Gong1, Wen-Zhe Fang 1, Zi-Cheng Bi1, Li-Hua Cheng1∗, Xin-Hua Xu1, Huan-Lin Chen2 1
2
Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, P.R.China
Department of Chemical and Biochemical Engineering, Zhejiang University, Zhejiang 310027, PR China.
Revision Submitted to Bioresource Technology June, 2015
∗
Corresponding author: Tel.(fax): +86-571-88982025 E-mail address: [email protected] (L.-H. Cheng). 1
ABSTRACT
Chlorella vulgaris, a marine microalgae strain adaptable to 0-50 g L-1 of salinity, was selected for studying the coupling system of saline wastewater treatment and lipid accumulation. The effect of total nitrogen (TN) concentration was investigated on algal growth, nutrients removal as well as lipid accumulation. The removal efficiencies of TN and total phosphorus (TP) were found to be 92.2-96.6% and over 99%, respectively, after a batch cultivation of 20 days. To illustrate the response of lipid accumulation to nutrients removal, C. vulgaris was further cultivated in the recycling experiment of tidal saline water within the photobioreactor. The lipid accumulation was triggered upon the almost depletion of nitrate (<5 mg L-1), till the final highest lipid content of 40%. The nitrogen conversion in the sequence of nitrate, nitrite, and then to ammonium in the effluents was finally integrated with previous discussions on metabolic pathways of algal cell under nitrogen deficiency.
Keywords: Nitrogen deficiency; Lipid accumulation; Saline wastewater; Microalgae; Chlorella vulgaris
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1. Introduction Saline wastewater，rich in both salt ( mainly NaCl) and nutrients，is often generated during the manufacture of food-processing, leather and petroleum industry, professionally known as produced waters (Lefebvre and Moletta, 2006). Utilization of seawater as part of water supply in situations where high quality water is unnecessary also promotes to generate large amounts of saline wastewaters. High salinity has been proved to strongly inhibit the metabolism of nitrifying bacteria and cause a failure to nutrients removal from saline wastewaters (Uygur and Kargi, 2004). In many studies, halophilic organisms were considered adaptable to treat saline wastewater in order to prevent pollutants (Duan et al., 2015; Zhang et al., 2014). Compared with screening of the organisms acclimated to saline wastewater, utilization of marine microalgae would be more manipulable and economical. Marine microalgae are usually efficient in removing nitrogen and phosphorus from wastewater, and have the ability to avoid contamination by biological pollutants such as bacteria, virus and zooplankton. Biological pollutants contamination has become a big constraint in massive microalgal cultivation, especially when cultured in wastewater (Wang et al., 2013; Yu et al., 2014). In our previous research, Scenedesmus obliquus was also found difficult to cultivate in the real municipal wastewater mainly due to the inevitable contamination of bacteria (Shen et al., 2015). To control infection and contamination of the biological pollutants, high salinity had been considered as one of solutions (Wang et al., 2013). Therefore, marine microalgae have the potential to play an important role in
3
saline municipal wastewater treatment. Meanwhile, microalgae can achieve potentially high yield of lipid and high economic value of harvested biomass if further produced for fertilizers, biogas, and so on (Cai et al., 2013). The coupling system of saline wastewater treatment and lipid production based on microalgae can be a promising solution to the water as well as energy crises (Li et al., 2010c). However, in the coupling system, how to ameliorate the performance of both nutrients removal and lipid accumulation is a central concern. To achieve high efficiency of nitrogen and phosphorus removal and to obtain high lipid productivity are the two purposes of the coupling system. To increase nutrients removal efficiency and lipid productivity simultaneously, Chlorella vulgaris, a strain of highly productive oleaginous marine microalgae is selected for studying the coupling system of lipid accumulation and saline wastewater treatment. Green unicellular microalgae have long been proposed as a potential renewable fuel source to produce biodiesel (Benemann et al., 1977). Chlorella vulgaris, when grown under nitrogen-sufficient conditions, produced biomass of approximately 0.17-0.18 g L-1 d-1, on a dry cell basis. Under nitrogen limitation, Chlorella vulgaris yielded total lipid content of 22%-63% by weight, mainly TAGs that are suitable for further production of biodiesel (Rodolfi et al., 2009; Stephenson et al., 2010). Meanwhile, Chlorella has been reported to be tolerant to various sewage effluent conditions and efficient in removing nutrients, such as nitrogen and phosphorus, from wastewater (Tuantet et al., 2014; Valderrama et al., 2002). Noting that Chlorella
4
vulgaris has not been utilized for nitrogen and phosphorus removal in saline wastewater. To induce the lipid accumulation in Chlorella vulgaris, the regulation strategy by nutrient limitations or deficiencies has been widely studied, including nitrogen, phosphate and sulfate, among which nitrogen deficiency is regarded as the most efficient approach. Li et al. (2010a) observed lipid accumulation as high as 30% of algal biomass for nitrogen limitation (initial TN of 2.5 mg L-1) by Scenedesmus sp. LX1. Stephenson et al. (2010) reported that leaving the algal cells in a medium containing 200 mg L-1 nitrate for 10 to 20 days would cause the natural depletion of nitrogen and thus enhance the lipid content, reaching up to 46% of TAG (triacylglycerol) in dry cell mass. However, upon what low level of nitrogen concentration the lipid would be triggered has not been reported in literature to our best knowledge. In this study, the effect of initial salt concentration on cell growth and lipid accumulation was first studied to investigate the potential of C. vulgaris for saline wastewater treatment. The nutrients removal and lipid accumulation, along with the nitrogen deficiency, were simultaneously investigated with different initial total nitrogen (TN) of 10 to 40 mg L-1. The recycling experiment within the photobioreactor was also investigated for a better understanding of lipid accumulation mechanisms. A real saline wastewater, the tidal saline water from Qiantang River (Zhejiang Province, China), was finally chosen to verify the feasibility of saline municipal wastewater treatment using C. vulgaris.
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2. Materials and methods 2.1 Algal species and municipal saline wastewater The marine microalgae Chlorella vulgaris, provided by the Key Laboratory of Marine Biotechnology, Ningbo University (Zhejiang, China), were cultivated. Before experiment, the strain was preserved in the artificial seawater medium (SM medium): 30 g L-1 instant sea salt, 2.5 mg L-1 FeSO47H2O, 1000 mg L-1 KNO3, 0.25 mg L-1 MnSO4, 10 mg L-1 KH2PO4, 6×10-3 mg L-1 Vitamin B1, 10 mg L-1 Na2EDTA, 5×10 -5 mg L-1 Vitamin B12. Wastewater environment was first provided by simulated municipal saline wastewater. NO3--N was used as the nitrogen source. In addition to nitrogen, the adapted artificial wastewater was prepared by dissolving the following chemicals: 13 mg L-1 KH2PO4 (3 mg L-1 PO43--P), 37.5 mg L-1 MgSO4·7H2O, 18 mg L-1 CaCl2·2H2O, 3 mg L-1 citric acid, 5 mg L-1 FeSO4·7H2O, 0.5 mg L-1 EDTA, 10 mg L-1 Na2CO3, 30 g L-1 sea salt and 1.0 mL L-1 A5+Co solution. The A5+Co solution contained 2.86 g L-1 H3BO3, 1.81 g L-1 MnCl2·H2O, 222 mg L-1 ZnSO4·7H2O, 79 mg L-1 CuSO4·5H2O, 390 mg L-1 Na2MoO4·2H2O, and 49 mg L-1 Co(NO3)2·6H2O. The initial pH was adjusted to 7.0-8.0. The tidal saline wastewater samples from Qiantang River were collected 1 hour before the tide coming, 1 hour and 2 hours after the tide during the tide period, with the average water conductivity of 2645 µS cm-1, 6217 µS cm-1 and 8810 µS cm-1, respectively. The TN concentrations ranged between 5.0 mg L-1 and 7.0 mg L-1, while
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the TP concentrations were about 0.30-0.35 mg L-1. 2.2 Experiment design The microalgae were first cultivated in a batch mode using artificial wastewater as nutrient supply. The experiments were conducted in 500 mL flasks containing 250 mL autoclaved artificial wastewater. The algal inoculums of 100 mL were centrifuged at 11180 × g for 10 min at 4°C, and the supernatant was discharged. The pellet of algal cells was then re-suspended into the artificial wastewater in a 500 mL flask. Nine flasks with an initial NaCl of 0-90 mg L-1 were prepared for the experiment to clarify the effect of initial NaCl concentration on cell growth and lipid content, while the initial TN and TP was 20 mg L-1 and 3 mg L-1, respectively. Four flasks with an initial TN of 10 mg L-1, 20 mg L-1, 30 mg L-1 and 40 mg L-1 were then prepared to study the effect of initial TN on algal growth, lipid accumulation and the removal of nitrogen and phosphorus. The initial TP and NaCl concentration for all the samples was kept at 3 mg L-1 and 30 g L-1. The second period was started with the same initial concentrations as the first one. The flasks of the two experiments mentioned above were then shaken under the continuous illumination of 40 µmol photons m-2 s-1 at 25°C. The experiments were conducted three times to verify the data reliability. In the recycling cultivation experiment, the culture system used was a cylindrical plexiglass photobioreactor with a working volume of 1 L (380 mm in height, 60 mm in diameter). Six fluorescent lamps were arranged around the photobioreactor to supply illumination. At the bottom of the reactor, a gas sparger was fixed and room air was
7
provided by a compressor. The experiment was conducted in three periods. After each period, the microalgae were retained in the reactor and the supernatant was discharged. Then the new wastewater with the initial nitrogen concentration of 20 mg L-1 was added into the reactor to begin a new period.
2.3 Analytical methods The biomass was evaluated by dry weight DW (g L-1), and its relationship with the optical density (OD) at 450 nm was referred as Eq. (1) (Fan et al., 2008): (1)
DW = 0.49 × OD450
The specific growth rate (µ，d-1) was used to describe the algal growth (Eq. (2)) (Jiang et al., 2011):
µ=
ln( DWt − DW0 ) t − t0
(2)
where DWt and DW0 were the dry weight at the end and the beginning of the exponential phase, respectively, and t-t0 was the duration time of the exponential phase. The lipid concentration L (g L-1) was measured by a high throughput Nile red method (Feng et al., 2013). The microalgae stained by Nile red (9-diethylamino-5H-benzo[a]phenoxazine-5-one) were put into water bath at 37°C for 10 minutes, and then determined by spectrofluorophotometer (Lengguang Tech. F96Pro). The relationship between fluorescence intensity F at 585 nm and L (g L-1) was determined using triolein as the lipid standard, as shown in Eq. (3):
F = 6 ×104 L + 25.56
(3) 8
The lipid content (% dry weight) was calculated from dividing L (g L-1) by dry weight DW (g L-1), as shown in Eq. (4):
Lipid content =
L × 100% DW
(4)
By dividing the difference in lipid (∆L) by the time interval (∆t, days), the lipid productivity (mg L-1 d-1) was obtained, as shown in Eq. (5).
Lipid productivity =
∆L ×1000 ∆t
(5)
For the nutrient removal analysis, the samples from flasks were centrifuged at 11180 × g for 10 min to determine TN, NO3-–N, NO2-–N, NH4+–N and TP in the supernatants. All the measurements were conducted according to Chinese State Standard Testing Methods (State Environmental Protection Administration, 2002).
3. Results and discussion To verify the feasibility of saline municipal wastewater treatment using Chlorella vulgaris, the effect of initial salt concentration on cell growth and lipid accumulation was first investigated. The effect of initial TN on the algal growth, nutrients removal and lipid accumulation was studied to illustrate the lipid accumulation in response to nitrogen stress.
3.1 Effect of salinity on algal growth and lipid accumulation Since the salt concentration of natural habitat of C. vulgaris is 34 g L-1, its growth
9
and lipid accumulation might be affected by salinity. The present study focused on the adaptation of C. vulgaris to variation of saline concentrations and their effect on the growth and lipid production. The flask cultivations were performed with several initial salt concentrations. As shown in Fig. 1, the dry weights of C. vulgaris remained stable at around 1.5 g L-1 in the salt concentration range of 0-50 g L-1. There was no significant effect of salinity on the algal growth between 0-50 g L-1. The dry weights under the conditions of 65 and 80 g L-1 were 0.9511 and 0.7908 g L-1, respectively. An apparent growth inhibition was observed when the salt concentration was higher than 50 g L-1. Consequently, the salt concentration lower than 50 g L-1 was considered appropriate for C. vulgaris to achieve high dry weight of biomass. The lipid content of biomass harvested at the end of cultivation under each condition was also shown in Fig.1. The lipid content of microalgae cultured without NaCl additional was only about 8%. The bar chart showed that there was a gradual increase in lipid content with the increase of salt concentration (below 50 g L-1). It indicated that the C. vulgaris was adaptable to 0-50 g L-1 of salinity with an increased production of lipid and might be suitable to treat saline wastewater with such a range of salinity.
3.2 Effect of initial TN on algal growth, lipid accumulation and the removal of nitrogen and phosphorus Fig. 2 reveals the algae growth, lipid accumulation and the removal of nitrogen and
10
phosphorus under different initial nitrogen concentrations in batch culture. The experiment was conducted three times to verify the data reliability. The second period was started with the same initial concentrations as the first one. As shown in Fig. 2(a), C. vulgaris grew faster when more nitrogen was initially supplied. After each period of cultivation, the algal dry weight reached about 1.8 g L-1 at the initial TN of 40 mg L-1. The TN and TP concentrations in the culture medium decreased rapidly with the growth of cells as shown in Fig. 2 (c) and (d). The concentration of TP decreased more rapidly than that of TN, regardless of the initial TN concentrations. Similar sudden decrease of TP at the beginning of experiment has been reported by Li et al. (2010a) that TP decreased from 1.3 mg L-1 to 0.8 mg L-1 during the first day of cultivation. The phosphorus removal from the synthetic wastewater was not due to precipitation as chelating chemicals were added to prevent precipitation occurring at high pH levels. This meant that the phosphorus had been removed due to a combination of growth, adsorption and luxury uptake of phosphorus (Powell et al., 2009). The removal efficiencies of TN and TP at the end of each batch were calculated to be 92.2~96.6% and nearly 100%, respectively, when the initial concentrations were controlled at TN of 10~40 mg L-1 and TP of 3 mg L-1, as shown in Fig. 2 (c) and (d), demonstrating its efficiency in removing nitrogen and phosphorus from saline wastewater. In the first period, after a batch cultivation of approximately 2, 7, 10 days, the TN was lower than 15 mg L-1 for the initial TN of 20, 30, 40 mg L-1, respectively. In
11
the second period, the TN was lower than 15 mg L-1 after a cultivation of approximately 1, 2, 3 days for the initial TN of 20, 30, 40 mg L-1, respectively. The TN removal efficiency in the second batch was apparently higher compared with that in the first batch which could be explained by the starvation period of about 10 days in the first batch. Fig. 2 showed that in the first period the highest removal rate of TN was 3.46 mg L-1 d-1 at the initial TN of 20 mg L-1, and the highest removal rate of TP was 2.26 mg L-1 d-1 at the initial TN of 40 mg L-1, indicating the high efficiency of the coupling system by Chlorella vulgaris in terms of nutrients removal. Although high removal efficiencies of nutrients were reported both with nitrate and ammonium as the nitrogen source, the removal rate of nutrients could be sensitive to the nitrogen source. Li et al. (2010b) reported a lower removal rate of TN and TP with ammonium as the nitrogen source, compared with nitrate as the nitrogen source. NH4+-N utilization produced H+ to cause acid pH, which would inhibit the algal growth during stable phase, thus reducing the removal rate of nutrients. The algal lipid content increased as shown in Fig. 2 (b) while the nitrogen was gradually removed as shown in Fig. 2 (c). By comparison of Fig.2 (b) and (c), the lipid accumulation was clearly observed at the almost depletion of the nitrogen. For example, in the first batch at the initial TN of 10 mg L-1, the nitrogen was almost depleted 2-4 days after inoculation. Induced by nitrogen stress, the lipid began to accumulate remarkably from the fifth day. Similar phenomena were observed at the initial TN of 20, 30, 40 mg L-1. After 20 days of batch cultivation in the first period, the final lipid
12
contents were measured to a range from 9.76% for initial TN of 40 mg L-1 to 33.07% for initial TN of 10 mg L-1, showing the lower the initial TN, the earlier nitrogen deficiency and the higher final lipid content. Compared with the first batch at the initial TN of 10 mg L-1, the nitrogen was consumed faster and depleted within one day in the second batch because of the 10 days starvation period in the first batch. Meanwhile, the lipid decreased in the first day and began to accumulate from the second day simultaneously. With a daily culture replacement of 1.0 L in a column aeration photobioreactor, Feng et al. (2011) reported C. vulgaris culture achieved the highest lipid content of 42%. Using Scenedesmus sp. cultivated at the initial TN of 2.5 mg L-1, Li et al. (2010a) obtained the lipid content of about 30%. The two batches of experiment and frequent monitoring of lipid revealed that high lipid content resulted from a lipid accumulation in response to TN concentration. In this work, the lipid was found to accumulate when the nitrogen concentration was lower than about 5 mg L-1 and the lipid content would decrease correspondingly with the nitrogen concentration exceeding 5 mg L-1, clearly showing that lipid was boosted at the critical nitrogen deficiency point. Although many studies had discussed the effect of nitrate limitation on fatty acid production by microalgae, our research focused on the the specific concentration of nitrate in response to the lipid accumulation by the cycling experiments, which has been rarely reported to the best of our knowledge. The experiment also demonstrated that cultivating the microalgae in the saline municipal wastewater using batch mode could not only further reduce the nutrients including
13
nitrogen and phosphorous, but also trigger the lipid accumulation without the further-assisted inducing methods, such as nitrogen-free environment (Stephenson et al., 2010) or strong irradiance (Hu et al., 2008). Stephenson et al. (2010) reported that rather than abruptly transfer the cells into a cultural medium lacking nitrogen, leaving the cells in a medium containing 200 mg L-1 nitrate for 10 to 20 days would enhance the lipid content (reached up to 46% of TAG in dry cell mass). The lipid content was found to accumulate more efficiently when the algal cells consumed the nitrogen in the medium naturally, which could be widely used in batch mode for boosting lipid content in biomass along with wastewater treatment.
3.3 Recycling microalgae in the photobioreactor As the shaking of flasks could not meet the need of C. vulgaris on carbon source, for example CO2, the cultivation of microalgae was further conducted in the photobioreactor with air aeration. The capabilities of algae growth, lipid accumulation and nitrogen removal by C. vulgaris were further attested in the photobioreactor. The recycling experiment within the photobioreactor was also investigated for a better understanding of lipid accumulation mechanisms. The algal biomass productivity, lipid productivity, TN and TP removal rates of each batch were investigated (Table 1 and Fig. 3). As shown in Fig. 3, the microalgae kept growing within all of the three periods. As shown in Table 1, the dry weight increased during operation, from 0.5929 g L-1 at the beginning to 1.038 g L-1 at the end. The
14
specific growth rate of the second batch was 1.51 d-1, which was much higher than that of the other two batches. As shown in Fig. 3, the microalgae entered the decline phase and the dry weight began to decrease after 5 days in the third batch. Since biomass increased continuously in the system during the treatment process, the removal efficiencies of nitrogen and phosphorus for the three batches increased at the same time. The removal rates of TN and TP in the third batch were 8.63 mg L-1 d-1 and 2.27 mg L-1 d-1, respectively, which were both the highest during the three batches. The nitrogen in the newly added wastewaters was instantly removed in less than 3 days at the beginning of each period. The lipid content varied against the nitrogen concentration. In response to the high nitrogen concentration in the newly added wastewater, the lipid content dropped a little. As soon as the nitrogen was almost depleted, the lipid content was boosted dramatically to a higher level. After the three periods, the lipid content achieved 40%, which was higher than that of the microalgae cultivated in the flasks. The maximum lipid productivity of the three periods were 20.15 mg L-1 d-1, 32.33 mg L-1 d-1 and 54.25 mg L-1 d-1, respectively. The highest lipid productivity in the third batch was due to the high nitrogen removal efficiency and the large quantity of biomass.
3.4 Nutrients removal from tidal saline water The tidal saline water from Qiantang River was further chosen to verify the feasibility of saline municipal wastewater treatment using C. vulgaris. Qiantang River
15
flows into the East China Sea via Hangzhou Bay and is well known for the large tidal bore. Water quality of Qiantang River is affected seriously by seawater intrusion into Qiantang River during tidal periods and the industrial wastewater and sewage excessive discharge in the upstream and downstream of Hangzhou City. Fig. 4 exhibited the variations in algal growth (a), lipid accumulation (b), nitrogen (c) and phosphorus (d) removal over three batch runs when cultivated within saline water. Fig. 4(a) and (b) exhibited the algal dry weight and lipid content over the three batches. The biomass dry weight increased gradually and reached about 0.34 g L-1 at the end of Batch 3, with slight difference between different runs of saline wastewater. The lipid content increased with increasing salinity, and reached 35% when cultured with saline water of 8810 µS cm-1. The results in Fig. 4 (c) and (d) demonstrated that C. vulgaris was able to remove total nitrogen and total phosphorus efficiently from saline water. The removal efficiency of TP was approximately 100% during the entire experiment which was not surprising as higher concentrations of TP were removed completely from artificial saline wastewater treatment experiments. The enhancement of lipid accumulation was not observed when cultured with water before the tide coming (2645 µS cm-1), even though TN had been removed below 5 mg L-1 (Fig. 4(b) and (c)). It indicated that an appropriate amount of salinity was essential for lipid accumulation of C. vulgaris under nitrogen deficiency, as already shown in Fig. 1. Our previous study proved it difficult to cultivate Scenedesmus obliquus in the real secondary municipal wastewater and the cells even disrupted after 7 days due to the contamination of bacteria (Shen et al., 2015). However, the marine C. vulgaris could remove nutrients and accumulate lipid efficiently within real tidal saline for three batches and remained active after 10 days in this study, in spite of the low 16
concentrations of nutrients within real tidal saline water. The difference showed the advantage of C. vulgaris to avoid contamination by bacteria and high salinity helped to control contamination by biological pollutants.
3.5 The potential mechanism of lipid accumulation triggered by nitrogen deficiency Although the lipid accumulation was clearly boosted at the point of nitrogen deficiency as shown in the Fig. 2 and Fig. 3, it was necessary to determine whether the deficiency of nitrate, ammonium, or other forms of nitrogen triggered lipid. The changes in NO3-–N, NO2-–N, NH4+–N for the initial TN of 20 mg L-1 were shown in Fig. 5. The NO3-–N was finally converted to NH4+–N in order to be utilized by C. vulgaris, during which the NO2-–N was produced. After the sixth day, the nitrate was almost depleted, while NH4 +–N remained at a relatively stable level. An increase of lipid was then observed after the sixth day. Therefore, although the nitrate could be converted to other forms of nitrogen after algal inoculation, the lipid accumulation was a response to the specific depletion of nitrate. Based on those from literature (Hu et al., 2008; Kliphuis et al., 2012; Li et al., 2012; Msanne et al., 2012), the potential mechanism diagram of lipid accumulation triggered by nitrogen stress was summarized as shown in Fig. 6. Ambient CO2, sun light and external nitrate/ammonium are normally utilized by microalgae as carbon, energy and nitrogen source, respectively, for maintaining life cycles including those involved in calvin cycle in chloroplast, tricarboxylic acid cycle (TCA cycle) and pentose phosphate pathway (PPP). A conversion of nitrogen from NO3-–N, to NO2-–N, and then to NH4+–N 17
had been observed in this work before they were transported into cytomembrane as shown in Fig. 6. The phenomenon was consistent with the result that microalgae prefer ammonium nitrogen to nitrate (Li et al., 2010b). When external nitrogen source was deficient for microalgal growth, the activity of calvin cycle involved in photosynthesis and electron transport was restrained as shown in blue lines while both the PPP and TCA cycles were strengthened as shown pink lines, resulting in low photosynthesis rate and a conversion from other components into lipid (Li et al., 2012; Msanne et al., 2012). Notice that Li et al. (2012) also found that carbohydrate was regarded as main carbon source for lipid synthesis as shown by the vertical arrow in Fig. 6, based on a decrease of intracellular carbohydrate content and the difference of relative genes’ transcription level between nitrogen-rich and nitrogen-free. This shift of metabolism to neutral lipids storage was an immediate response of microalgae to the stress from the cultural environment and served as a reserve for rebuilding the cell after the stress (Rodolfi et al., 2009; Roessler, 1990). Although the conversion of carbohydrate, protein and Chlorophyll through TCA cycle for further lipid synthesis has been reported, more information is still required for the metabolic pathways and functional genes involved in this process.
4. Conclusions The marine microalgae strain C. vulgaris was successfully used to treat saline wastewater including the tidal saline water. The lipid accumulation was triggered at the
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point of nitrate concentration below 5 mg L-1. The lipid content increased up to 40% and the maximum lipid productivity was 54.0 mg L-1 d-1 at the end of the recycling system in the photobioreactor. This study also showed the feasibility of C. vulgaris to remove TN and TP efficiently from real saline wastewater owing to its ability to avoid contamination of bacteria.
Acknowledgement This work is based upon research supported by Important National Science & Technology Specific Projects (2014ZX07101-012-04), the National Natural Science Foundation of China (No. 21106130, No. 21276221) and the Doctoral Discipline Foundation for Young Teachers in the Higher Education Institutions of Ministry of Education (201101011120074). We gratefully acknowledge to the Key Laboratory of Marine Biotechnology, Ningbo University (Zhejiang, China) for generously presenting Chlorella vulgaris.
References
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Table captions
Table 1. Algal biomass production, lipid accumulation and nutrients removal of the three batches cultivation with artificial wastewater in photobioreactors
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Figure captions Fig. 1 Effect of initial salinity on the algal growth and the lipid accumulation. (initial nitrate concentration: 20 mg L-1, initial phosphorus concentration: 3 mg L-1, light intensity: 40 µmol photons m-2 s-1, temperature: 25 °C) Fig. 2 Effect of initial TN concentration on the algal growth within two batches (a), the lipid accumulation (b), the nitrogen (c) and the phosphorus (d) removal. (initial NaCl concentration: 30 g L-1, initial phosphorus concentration: 3 mg L-1, light intensity: 40 µmol photons m-2 s-1, temperature: 25 °C) Fig. 3 Changes in algal growth, nitrogen removal and lipid accumulation in biomass over three batch runs in the photobioreactor. (initial nitrogen concentration: 20 mg L-1, initial phosphorus concentration: 3 mg L-1, light intensity: 40 µmol photons m-2 s-1, temperature: 25 °C) Fig. 4 Changes in algal growth (a), lipid accumulation (b), nitrogen (c) and phosphorus (d) removal over three batch runs when cultivated with tidal saline water. Fig. 5 Time course of NO3--N, NO2--N, NH4+-N concentrations for the initial TN of 20 mg L-1. Fig. 6 Schematic of lipid accumulation triggered by nitrogen deficiency. (TCA cycle, PPP, GAP and G6P refer to tricarboxylic acid cycle, pentose phosphate pathway, glyceraldehyde 3-phosphate and glucose 6-phosphate, respectively.)
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Table 1 Algal biomass production, lipid accumulation and nutrients removal of the three batches cultivation with artificial wastewater in photobioreactors Photobioreactor Stage 1
Stage 2
Stage 3
Max. biomass concentration (g L-1) 0.5929
0.9070
1.0380
Specific growth rate (d-1)
1.09
1.51
1.35
Lipid content (%)
24.1
31.4
40.1
Lipid productivity (mg L-1 d-1)
20.15
32.33
54.25
Removal rate of TP (mg L-1 d-1)
1.32
2.09
2.27
Removal rate of TN (mg L-1 d-1)
7.61
17.29
8.63
25
Highlights:
1. C. vulgaris was found suitable for saline wastewater treatment.
2. Salinity was essential for lipid accumulation of C. vulgaris.
3. Lipid content was boosted at point of nitrate deficiency by Nile red method.
4. High nutrient removal efficiencies could be obtained by recycling cultivation mode.
5. Pollutants in a real tidal saline water were efficiently utilized by microalgae.
26
27
a
b
c
d
28
29
a
b
c
d
30
31
32