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The ΔF/Fm′-guided supply of nitrogen in culture medium facilitates sustainable production of TAG in Nannochloropsis oceanica IMET1
© The Author(s) 2018
Received: 5 December 2017
Accepted: 12 June 2018
Published: 20 June 2018
Triacylglycerol (TAG) from photosynthetic microalgae is a sustainable feedstock for biodiesel production. Physiological stress triggers microalgal TAG accumulation. However excessive physiological stress will impair the photosynthesis system seriously thus decreasing TAG productivity because of the low biomass production. Hence, it is critical to quantitatively and timely monitor the degree of the stress while the microalgal cells growing so that the optimal TAG productivity can be obtained.
The lack of an on-line monitored indicator has limited our ability to gain knowledge of cellular “health status” information regarding high TAG productivity. Therefore, to monitor the degree of nitrogen stress of the cells, we investigated the correlation between the photosynthetic system II (PS II) quantum yield and the degree of stress based on the high relevancy between photosynthetic reduction and nitrogen stress-induced TAG accumulation in microalgal cells. ΔF/Fm′, which is the chlorophyll fluorescence parameter that reflects the effective capability of PS II, was identified to be a critical factor to indicate the degree of stress of the cells. In addition, the concept of a nitrogen stress index has been defined to quantify the degree of stress. Based on this index and by monitoring ΔF/Fm′ and guiding the supply of nitrogen in culture medium to maintain a stable degree of stress, a stable and efficient semi-continuous process for TAG production has been established.
The results indicate that the semi-continuous cultivation process with a controlled degree of stress by monitoring the ΔF/Fm′ indicator will have a significant impact on microalgal TAG production, especially for the outdoor controllable cultivation of microalgae on a large scale.
Photosynthetic microalgae have received increasing attention as the most promising biofuel feedstock as humans are facing increasing problems related to climate and fossil energy [1–3]. Microalgae can use light energy and CO2 to produce energy-storage compounds such as triacylglycerol (TAG), which is the precursor of biodiesel . Physiological stress is usually applied to microalgal cultures to trigger TAG accumulation. When confronted with physiological stress, such as nitrogen stress (N-stress), microalgal cells make adjustments such as enhancing the energy-storage compounds (e.g., TAG) accumulation, to acclimate to unfavourable conditions. However, the physiological stress affects both photosynthesis and C-storage mechanism. When under excessive physiological stress, the photosynthetic efficiency progressively decreases and the cell growth is significantly diminished. Therefore, it is quite challenging to achieve maximum TAG productivity by balancing the TAG content and high productivity of microalgal biomass because TAG production largely relies on stress conditions such as nitrogen stress (N-stress) and high irradiance etc. .
N-stress is the most effective means to trigger TAG accumulation [4, 6, 7]. When subjected to N-stress, in addition to enhanced TAG accumulation, protein synthesis in microalgal cells is affected as well. Protein synthesis in microalgae is immediately suppressed upon nitrogen shortage, which mostly hinders the protein turnover of photosynthetic apparatus, especially the photosystem (PS) II D1 reaction centre protein . This will lead to a decline in the photosynthetic electron transport rate (ETR) and, consequently, a reduction in photochemical energy conversion [8, 9]. Moreover, limited nitrogen supply causes impairment of photosynthetic CO2 fixation by degradation of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) for the recycling of nitrogen. Limitation of CO2 fixation then decreases the consumption of ATP and NADPH and leads to an excess of NADPH and electrons . The sufficient supply of NADPH is essential for TAG accumulation , whereas excessive electrons will lead to the formation of reactive oxygen species (ROS), which exposes microalgae to oxidative stress and is also believed to be a signal trigger for TAG formation [11–13]. In addition, the remodelling of the photosynthetic membranes caused by N-stress also contributes to a considerable fraction of TAG production by providing a fatty acid acyl moiety [5, 14, 15]. Therefore, it is closely linked to photosynthetic reduction and N-stress-induced TAG accumulation in microalgal cells. Therefore, it is very important to make systematic research on it.
Photosynthesis is a coordinated physiological process that exclusively provides both energy and the material foundation for photoautotrophic microalgae . Therefore, it could be considered the most important cellular metabolism in algae. With no exception, photosynthesis provides the energy as well as fixes the carbon used for TAG synthesis in photoautotrophic microalgae. However, as stated above, TAG accumulation is inevitably accompanied by photosynthetic reduction under excessive stress in photoautotrophic oleaginous microalgae, which means that excessive stress could cause inhibition of growth and reduce biomass and overall TAG yield. Optimal TAG productivity can be achieved only if the photosynthetic performance is properly maintained. Therefore, quantitative and timely monitoring of the stress status of the subjected microalgal cells is vital so that the optimal TAG productivity can be attained in time. Because of the tight relationship between photosynthesis and TAG synthesis, monitoring of the photosynthetic performance should be a plausible method.
Chlorophyll fluorescence analysis is a powerful tool for the study of photosynthesis in both plants and algae [17–19]. It allows a non-invasive and nearly instantaneous measurement of performance in photosynthetic light capture and electron transport . The PS II quantum yield, which is a chlorophyll fluorescence parameter that measures the proportion of the light absorbed by chlorophyll related to PS II and is used in photochemistry, provides an estimation of the linear ETR and hence the PS II performance [18, 19]. It is widely used as the indicator to assess nutrient limitations in situ in microalgae [9, 20, 21]. However, to the best of our knowledge, little research has been conducted hitherto to apply this fluorescence parameter for the control of TAG production. In our previous work, a decreased PS II quantum yield was found to be highly related to the degree of N-stress. In both Tetraselmis subcordiformis and Isochrysis zhangjiangensis, critical values of chlorophyll fluorescence existed where the maximum productivities of energy storage substances, such as carbohydrates, were obtained [22, 23]. Therefore, in this work, we proposed the use of this fluorescence parameter as an N-stress indicator to monitor the stress degree and TAG production during cultivation by controlling the nitrogen supply strategy. To obtain an efficient and stable production of lipid from Nannochloropsis oceanica IMET1, we established a reproducible TAG production process by monitoring photosynthetic activity to maintain a constant degree of N-stress under semi-continuous cultivation.
Strain and Cultivation conditions
Nannochloropsis oceanica IMET1 from the University of Maryland Biotechnology Institute was cultivated in seawater with modified F/2 medium at the ambient temperature of 25 ± 1 °C. In the present study, a 500-mL bubble column bioreactor (5 cm in diameter) was used with the air flow at 100 mL/min (2% CO2, v/v) filtered by a 0.22-μm membrane as described by Pan et al. . Additionally, 140 μmol/m2 s cool white fluorescent lights were provided from one side of the bioreactor under a 14-h/10-h light/dark cycle. After pre-culture in the bioreactor with sufficient nutrient elements until the cells reached exponential growth phase, the cells were inoculated into new bioreactors at an initial biomass concentration of ~ 0.18 mg/mL (dry weight, DW).
For the batch culture, the initial N amount was 15 mg/L in the medium, and no additional N was added into the medium until harvest. For the semi-continuous cultivation, the inoculum was the N-stressed cells from the batch. During cultivation, the fluorescence parameter ΔF/Fm′ was monitored. During the semi-continuous, ΔF/Fm′ was used as an indicator of the degree of N-stress. When ΔF/Fm′ reached the predetermined level, which was determined in batch cultivation as the monitoring point, harvest and dilution were conducted to start a new cycle of cultivation. During the dilution, the relative amount of fresh medium replenished to the system was defined as the “renewal rate”. For each cycle during the same semi-continuous cultivation process, the initial biomass concentration and supplementary N amount were the same.
Results and discussion
Verification of ΔF/F m′ as an N-stress indicator for TAG production
When comparing all of the parameters, intracellular N is a critical parameter to trigger the initiation of TAG accumulation. There were studies that made efforts on the determining the proper N-stress and emphasized the importance of accurate control of intracellular N when to optimize the lipid productivity [30, 31]. However, the lack of a fast detection method for intracellular N limits its application as an online indicator to control microalgal cells at a proper degree of N-stress and to balance cell growth and TAG accumulation. Among the measured parameters, ΔF/Fm′ is a promising indicator of N-stress in microalgae due to the high correlation between intracellular N and ΔF/Fm′. By investigating the growth of N. oceanica IMET1 under N-stress, it was found that during the period of day 3 to day 6 when the highest TAG productivity under N-stress was obtained, there was a high correlation between intracellular N and ΔF/Fm′. The correlation coefficient between intracellular N and ΔF/Fm′ reached 0.97 (y = 0.1083x + 0.1258, where R2 = 0.9713, y refers to ΔF/Fm′, and x refers to intracellular N). In the other species of microalgae, a correlation also exists between intracellular N and ΔF/Fm′. For T. subcordiformis and I. zhangjiangensis, the correlations were y = 0.0741x + 0.151 (R2 = 0.9779) and y = 0.168x + 0.1597 (R2 = 0.9302), respectively (unpublished data). In short, it is not a special case where the ΔF/Fm′ and intracellular N synchronously change while cells are under the N-stress condition. The great advantages of ΔF/Fm′ are its characteristic of in situ and rapid measurement. In addition, the photosystem (whose activity ΔF/Fm′ reflects) plays the role of the hub in microalgal metabolism. Therefore, we propose monitoring the degree of N-stress using ΔF/Fm′ as an on-line indicator and then making the unsteady status of high TAG productivity reproducible to realize continuous TAG production.
Determination of the proper degree of N-stress of microalgal cells for TAG production
Stable reproduction of TAG under semi-continuous cultivation by monitoring ΔF/F m ′
Operating conditions of five semi-continuous experiments
Initial N concentration (mg/L)
To keep the culture conditions consistent with that of the batch cultivation in “Determination of the proper degree of N-stress of microalgal cells for TAG production” section, in experiment a, the initial N concentration was set to 24 mg/L. The initial cell density and total amount of N, including the intracellular N and free N in the medium, were regulated to the same level as that in the batch cultivation. It should be noted that cycle 1 was started at day 0 and ended at day 4; at the same time, day 4 was also the beginning point of cycle 2, which ended at day 8. The intracellular N at day 0, day 4 and day 8 coincided with each other as shown in Fig. 3a-1. The NSIs were maintained as almost equal, with values of 0.72, 0.71 and 0.70, respectively. The DW and TAG content also showed the same trend between cycles. The results indicated the successful repeat of the proper N-stressed cells defined as day4-cell with an NSI of 0.72 in “Determination of the proper degree of N-stress of microalgal cells for TAG production” section.
Experiments b and c were performed to investigate the effects of the initial N concentration in medium on TAG productivity. As shown in Fig. 4b, c, the TAG productivity of each cycle during experiments b and c were lower than that during experiment a. It is common sense that the DW increment of each cycle is dependent on the total N in the medium. For example, regarding cycle 2 in experiment a, the intracellular N at the end point was set to be equal to the initial intracellular N of 3.82%, and the total N amount added into the medium was fixed to be 22.9 mg/L; thus, theoretically, the DW increment of cycle 2 should be 0.60 mg/mL (the amount of N added into the medium each time divided by the initial intracellular N). Practically, the actual DW increment was 0.65 mg/mL in accordance with the theoretical value. In other words, when the degree of N-stress of the cells at the end point was fixed, which indicated that the intracellular N was set to be at a fixed value, the total N amount added into the medium would decide the absolute increment of DW. Therefore, it was the cell growth rate that actually determined the TAG productivity of each cycle. Additionally, the DW increase rates in experiments b and c were 0.13 and 0.09 mg/mL day, respectively, and were lower than that of experiment a, which was 0.18 mg/mL day. The lower cell growth rate was caused by the lower photosynthetic activity of the cells. In semi-continuous cultivation, the cells used as inoculum were N-stressed with the “sub-health” status. After resupplying the N nutrient, the stressed cells started to recover towards the “health” status. The photosynthetic activity of the cells was recovered as one of the important physiological features. As shown in Fig. 4b-1, c-1, the ΔF/Fm' recovered to the peak of 0.55 and 0.52 during each cycle in experiment b and c after N replenishment and recovered to 0.58 in experiment a. Obviously, resupplying the N amount has a large effect on the level of photosynthetic activity recovery. In experiments b and c, the cells did not recover to the health status as in experiment a, leading to the lower cell growth and lower TAG productivity.
Compared to the healthy cells with sufficient nutrition, there was an obvious reduction in the photosynthesis activity of the proper N-stressed cells as shown in Fig. 1a, which hindered the cell growth. However, the rate of TAG accumulation of the stressed cells received a tremendous boost instead and remedied the sacrifice of cell growth. Hence, via the semi-continuous cultivation with maintenance of the degree of N-stress by controlling the N supply strategy, stable, sustainable TAG production was achieved.
Taken together, the data indicate that ΔF/Fm′ is a rapid and in situ N-stress indicator and could precisely guide the control of the degree of N-stress during the cultivation of microalgae for efficient and stable TAG production.
It is vital to quantify the degree of N-stress when it is applied to obtain maximum TAG productivity during microalgal cultivation. The concept of the nitrogen stress index, NSI, was defined. The photosynthetic activity parameter of microalgae, ΔF/Fm', was identified as a perfect online indicator for the degree of N-stress of the cells. Based on NSI, a novel semi-continuous cultivation strategy of precise N-stress control by the accurate monitoring of ΔF/Fm' as an N-stress indicator was established for stable and efficient TAG production. The renewal rate and initial N concentration were optimized as 0.6 and 24 mg/mL, respectively. This new cultivation strategy provides significant guidance for outdoor microaglal cultivation in industrial applications and controlled indoor cultivation.
SX, JL, CY and XC designed the research. SX, JL and CY wrote the paper. SX, JL and XC analyzed the data. JL, YM and PW performed the research and provided technical support. All authors read and approved the final manuscript.
All authors acknowledge the support by the National Nature Science Foundation of China and the Ministry of Science and Technology of China. The authors thank Prof. Qiang Hu from the Institute of Hydrobiology, Chinese Academy of Sciences and for valuable comments on this study.
The authors declare that they have no competing interests.
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