Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. 2018, 66, 10777−10787
Phosphorus Enhances Photosynthetic Storage Starch Production in a Green Microalga (Chlorophyta) Tetraselmis subcordiformis in Nitrogen Starvation Conditions Changhong Yao,*,† Junpeng Jiang,‡,§ Xupeng Cao,‡ Yinghui Liu,‡ Song Xue,*,‡ and Yongkui Zhang†
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†
Department of Pharmaceutical & Biological Engineering, School of Chemical Engineering, Sichuan University, Chengdu, Sichuan 610065, China ‡ Marine Bioengineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China § University of Chinese Academy of Sciences, Beijing 100039, China S Supporting Information *
ABSTRACT: Microalgae are potential starch producers as alternatives to agricultural crops. This study disclosed the effects and mechanism of phosphorus availability exerted on storage starch production in a starch-producing microalga Tetraselmis subcordiformis in nitrogen starvation conditions. Excessive phosphorus supply facilitated starch production, which differed from the conventional cognition that phosphorus would inhibit transitory starch biosynthesis in plants. Phosphorus enhanced energy utilization efficiency for biomass and storage starch production. ADP-glucose pyrophosphorylase (AGPase), conventionally known to be critical for starch biosynthesis, was negatively correlated to storage starch biosynthesis. Excessive phosphorus supply maintained large cell volumes, enhanced activities of starch phosphorylases (SPs) along with branching enzymes and isoamylases, and increased phosphoenolpyruvate and trehalose-6-phosphate levels to alleviate the inhibition of high phosphate availability to AGPase, all of which improved starch production. This work highlighted the importance of phosphorus in the production of microalgal starch and provided further evidence for the SP-based storage starch biosynthesis pathway. KEYWORDS: microalgae, storage starch, phosphorus supply, ADP-glucose pyrophosphorylase, starch phosphorylase
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INTRODUCTION Starch serves as the primary carbon sink for most photosynthetic plants and microalgae.1 It not only plays important roles in agriculture as foods but also is used as a sustainable feedstock for biofuels and biobased chemicals production.2 Microalgae, a kind of photosynthetic microorganism, are able to capture CO2 and sequentially store large amounts of starch intracellularly.3,4 Due to the very similar structure and characteristics of algal starch, plus the high photosynthetic efficiency, fast growth rate, and flexible cultivation modes of microalgae, microalgal starch is recently considered to be a promising alternative to crop-based starch.5 Microalgal starch accumulation is usually induced under stressful conditions, among which nitrogen starvation is in general the most effective trigger.6,7 Unlike plants that photosynthetically synthesize transitory starch in source tissues (e.g., leaves) and storage starch in sink tissues (e.g., seeds and tubers),8 unicellular microalgae, which have no tissue differentiation, synthesize both transitory and storage starch at the same location of the cells. It has been proposed that nitrogen starvation-induced starch in microalgae can be regarded as the storage starch.9 In terms of these aspects, the storage starch biosynthesis in microalgae should be different from the plants. However, very limited information is available on this issue. There is only a partial picture of the microalgal storage starch biosynthesis in the model microalga Chlamydomonas reinhardtii.9−11 © 2018 American Chemical Society
Phosphorus is an integral component of nucleic acids and phospholipids, an essential modifier of protein function, and is fundamental to the cell’s energy currency.12 However, phosphorus supply is usually regarded to be unbeneficial to starch accumulation mainly for the following two reasons.8 On the one hand, high level of phosphate in cytosol will accelerate the export of triose-phosphate (e.g., glyceraldehyde 3phosphate and dihydroxyacetone phosphate) from chloroplast to cytoplast and import of phosphate from cytoplast into chloroplast through triose phosphate/phosphate translocator (TPT), resulting in the loss of substrates in the chloroplast for starch biosynthesis. On the other hand, the consequential high concentration of phosphate in chloroplast will exert inhibitory effects on ADP-glucose pyrophosphorylase (AGPase), a critical enzyme involved in the committed step for starch biosynthesis. As a consequence, phosphorus deficiency generally results in increased transitory starch accumulation in leaves (reviewed by MacNeill et al.8). In contrast, phosphorus has been recently reported to facilitate storage starch accumulation in wheat grain13 and potato tubers,14 suggesting that phosphorus played distinct roles in the biosynthesis of these two types of starch. In microalgae, a reverse correlation between phosphorus availability and starch accumulation has been documented in Received: September 3, 2018 Accepted: October 1, 2018 Published: October 1, 2018 10777
DOI: 10.1021/acs.jafc.8b04798 J. Agric. Food Chem. 2018, 66, 10777−10787
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Journal of Agricultural and Food Chemistry Pseudochlorococcum,15 Chlorella,16 and Arthrospira.17 However, recently, a positive effect of phosphorus on starch accumulation has been observed in Scenedesmus obliquus.18 These findings evoke a reconsideration of the relationship between phosphorus availability and storage starch biosynthesis, and the existing knowledge of starch biosynthesis pathway as well as its regulation mechanism may need to be further improved, especially in nonmodel microalgae. To verify the specific role of phosphorus in the storage starch accumulation in microalgae, the present study investigated the effects of phosphorus availability on the nitrogen starvation-induced starch accumulation in a starchproducing microalga Tetraselmis subcordiformis. The phosphorus assimilation and intracellular free phosphate levels were tracked to clarify the phosphorus accessibility. Meanwhile, the activities of key enzymes involved in starch biosynthesis [AGPase, starch phosphorylase (SP), starch synthase (SS), branching enzyme (BE), isoamylase (ISA)] and degradation [amylase (AMY)] were also determined. In addition, the variations of metabolites in relation to starch biosynthesis and central carbon metabolism were further measured to disclose the reasons for the effects of phosphorus exerted on starch accumulation in this microalga.
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optical microscope (NI-U, Nikon, Japan). The length and width of three typical representative living cells in each culture were measured using ImageJ (https://imagej.nih.gov/ij/). The cell geometry was regarded as oblate ellipsoid for volume calculation. The starch content in algal cells was quantified according to Brányiková, et al.4 with minor modifications. Briefly, samples of 1 mL of the algal suspension were harvested by centrifuging at 4 000g for 5 min, and the pellets were resuspended in 1 mL of 30% perchloric acid, stirred for 15 min at 25 °C, and centrifuged. This procedure was repeated three times. The extracts were combined and made up to 6 mL. Thereafter, 0.5 mL of the extract was mixed with 0.5 mL of H2O, and 5 mL of anthrone solution [2 g of anthrone in 1 L of 72% (v/v) H2SO4] was added and stirred. The mixture was kept in a water bath at 100 °C for 8 min. It was then cooled to 20 °C, and the absorbance was measured at 621 nm. The blank assay for starch determination was carried out by adding equal amounts of reagents to a microalgaefree sample. Glucose was used as the standards for calibration. The values measured for glucose were multiplied by 0.9 to obtain the levels of starch. Starch content (Cs, % of DW) was calibrated by subtracting the starch content from blank assay. Starch productivity (Ps, g/L/d) was calculated as follows: Ps =
(2)
where Cst and Cs0 are the starch content at culture times t and 0, respectively. Phosphate Analysis. The extracellular phosphate (ExPi) and intracellular phosphate (InPi) levels were determined according to Yao et al.20 Briefly, a 1 mL aliquot of algal culture was centrifuged at 4 000g for 5 min and the supernatant was used for ExPi analysis. For InPi measurement, an equal aliquot of algal culture was sonicated before centrifugation to obtain the total free phosphate (TFP). The amount of phosphate was determined by a molybdate-based colorimetric assay.21 The InPi concentration was estimated as follows:
MATERIALS AND METHODS
Algal Strain and Culture Conditions. T. subcordiformis FACHB-1751 was isolated from the Huanghai Sea near Dalian, Liaoning Province, China, and maintained by the Freshwater Algae Culture Collection of the Institute of Hydrobiology (FACHB collection), Chinese Academy of Sciences. The microalgae were previously cultivated in natural seawater by adding nutrients, as described by Yao et al.19 Algal cells were harvested during the late exponential phase and washed twice with nitrogen and phosphorus-free artificial seawater (ASW-N-P). The ASW-N contained (per liter): 27.0 g of NaCl, 0.075 g of MgSO4· 7H2O, 0.036 g of CaCl2·2H2O, 0.04 g of NaHCO3, 0.89 g of K2SO4, 0.8 mg of FeCl3, 0.4 mg of MnCl2·4H2O, 33.6 mg of H3BO3, 45.0 mg of ethylenediaminetetraacetic acid disodium salt, 0.21 mg of ZnCl2, 0.2 mg of CoCl2·6H2O, 0.09 mg of (NH4)6Mo7O24·4H2O, and 0.137 mg of CuCl2·2H2O. The pH was adjusted to 6.5 with HCl. The washed cells were inoculated with 1.5 × 106 cells mL−1 in ASW-N-P with the addition of NaH2PO4 to final concentrations of 0, 3, and 9 mM, respectively. The cells were cultivated in a 600 mL glass air bubble column photobioreactor (50 mm diameter, 400 mm height) with a working volume of 500 mL and an aeration of 0.4 vvm with air containing 2% CO2 at 25 ± 2 °C as described by Yao et al.19 The cultures were continuously illuminated from one side with cool white fluorescent lamps that provided an incident light intensity of 150 μmol/m/s measured by a photosynthetically active radiation (PAR) detector (Optometer P9710 with PAR detector 3701, Gigahertz Optik Corporation, Germany). The data shown in the figures as points or columns with error bars are means and standard deviations (SD) of three biological replicates. Growth and Starch Measurement. The cell growth was determined as the optical density of the culture at 750 nm on a spectrophotometer (UV−vis V-530, Jasco, Japan). The cell dry weight (DW) was determined gravimetrically according to Yao et al.19 Biomass productivity (Pb, g/L/d) was calculated as follows: DWt − DW0 Pb = t
DWtCst − DW0Cs0 t
InPi =
TFP − ExPi OD750 × a × V
(3)
where a and V represented the OD750-cell density conversion factor (a = 202.7 × 104 cells/mL) and cell volume, respectively. Photosynthetic Performance Analysis. The photosynthetic performance with regard to the photosystem II (PS II) operating efficiency (ΔF/Fm′) and nonphotochemical quenching (qN) were evaluated with chlorophyll fluorescence determined using a chlorophyll fluorometer (Water-PAM WALZ, Germany). Briefly, algal cells were adapted in the dark for 10 min, and a measuring light (ML, 15 μmol m−2 s−1) was applied to obtain the minimum initial fluorescence F0, after which a program named “light inductive curve” was run, where a saturating light pulse (SP, 0.6 s, 1416 μmol/m2/s) was immediately applied to evaluate the maximum fluorescence at dark-adapted state Fm. Then the maximum fluorescence at lightadapted state Fm′ was measured by applying a saturating light pulse (SP, 0.6 s, 1416 μmol/m2/s) after the cells were continuously illuminated for 4 min under actinic light (AL, 481 μmol/m2/s), which also generated a light-adapted fluorescence F′. The measurement applied an excitation at wavelength of 660 nm, and the fluorescence was detected at wavelengths 0.1) was observed between different phosphorus treatments at each cultivation time.
nitrogen starvation prolonged, and no significant difference could be detected among these three phosphorus-level treatments tested. The activity of AGPase decreased to approximately half of the original level within 24 h. Since starch biosynthesis was stimulated under nitrogen starvation, as demonstrated in Figure 1d, the pivotal role of AGPase in starch biosynthesis seemed controversial herein. In fact, some omics data dissecting metabolic regulation of microalgae (e.g., Neochloris oleoabundans and Chlamydomonas reinhardtii) in response to nitrogen starvation also presented a downregulation of AGPase at transcription or translation levels, although starch biosynthesis occurred in cells at that time.38−40 Considering that the starch biosynthesis in T. subcordiformis under nitrogen starvation was not affected in the presence of high phosphate level intracellularly, and the phosphate even facilitated starch accumulation during the late phase of nitrogen starvation (Figure 1d and Figure 3b), it could be deduced that AGPase should not be a dominator for storage starch biosynthesis in T. subcordiformis. As for SP, it has been previously reported that T. subcordiformis has four SP isoforms (TsSP1-TsSP4), among which TsSP4 was considered to locate in the plastid via sequence alignments.23 It was evident from Figure 5b that the activity of TsSP2/3 and TsSP4 exhibited an overall increase with the extension of cultivation time under nitrogen starvation in both phosphorus-deprived (0 mM) and phosphorus replete/ superfluous cultures (3 mM/9 mM). Similarly, the phosphorylase activities were reported to increase with the accumulation of glycogen in yeast under nitrogen starvation conditions,41 which was consistent with the present observation in T. subcordiformis. In addition, the SP activity was clearly detected first only in the culture with the highest phosphorus availability (9 mM) at 6 h, after which SP activity was detectable in all the
Figure 4. Variations of photosystem II operating efficiency (ΔF/Fm′, (a) and nonphotochemical quenching (qN, (b) in Tetraselmis subcordiformis under nitrogen starvation in response to different phosphorus availabilities (means ± SD, n = 3). “++” denoted the significant differences (p < 0.1) observed between cultures of 3 mM and 0 mM as well as 9 mM and 0 mM; “+++” denoted the significant differences (p < 0.1) observed between each treatment.
cultures, which in fact did not exhibit any significant difference among the series of phosphorus supply tested at 48 h. In contrast, qN showed an overall increase, and the phosphorusenriched cultures (3 mM and 9 mM) displayed less pronounced rise of qN than that in the phosphorus-deprived (0 mM) one (Figure 4b). Typically, qN in the phosphorusenriched cultures (3 mM and 9 mM) remained at 0.18 within 24 h, whereas it increased greatly to 0.35 in the phosphorusdeprived (0 mM) one. The decrease in photosynthetic activity under nitrogen starvation was due to the shortage of nitrogen for protein synthesis (Figure S2), while the increase in heat dissipation is a common strategy for microalgae to protect themselves from severe photodamage when exposed to stress conditions.36,37 The identical photosynthesis ability but less heat dissipation (i.e., less energy loss) in phosphorus-enriched cells along with the improved cell growth (Figure 1a) relative to the phosphorus-deprived ones suggested that excessive intracellular phosphate mediated some of the assimilated energy to be utilized for biomass and starch accumulation instead of being dissipated as heat. This also meant that phosphorus supply could enhance energy utilization efficiency rather than improving photosynthetic energy assimilation in T. subcordiformis under nitrogen starvation conditions. 10782
DOI: 10.1021/acs.jafc.8b04798 J. Agric. Food Chem. 2018, 66, 10777−10787
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Journal of Agricultural and Food Chemistry cultures at 24 h, with the culture of 3 mM phosphorus showing the highest activity for TsSP4. At 48 h, the activity of TsSP4 ascended with the increase of phosphorus availability. Overall, the performance of SP, especially plastidial TsSP4, appeared to be activated by phosphorus supply. SP catalyzes the reversible conversion of α-1,4-glucan and inorganic phosphate (Pi) into glucose-1-phosphate (Glc-1-P) and a glucan-(one glucosylresidue), the direction of which depends on the Pi/Glc-1-P ratio that the enzyme was exposed to.42 High Pi generally favors phosphorolysis rather than synthesis (glucan elongation) reaction. Interestingly, in T. subcordiformis, TsSP4 had been demonstrated to be able to elongate maltooligosacchride (MOS) even in the presence of high concentration of phosphate (10 mM) alone.23 TsSP4 had been shown to thermodynamically prefer synthetic directions for the elongation of MOSs, and the activity in the synthesis direction always exhibited 2−3-fold of that in the phosphorolytic direction.23 Furthermore, the preference of TsSP4 for synthetic directions was more pronounced with the increase of chain length of MOSs.23 Therefore, it appeared that once the phosphate triggered the phosphorolysis of MOS which simultaneously released Glc-1-P, TsSP4 could immediately catalyze the glucan elongation reaction with the MOS and released Glc-1-P as substrates, leading to the formation of longer MOSs, and the elongation should be further facilitated when more long-chain MOSs were produced. In the present study, although the phosphate concentration in the chloroplast might be relatively high when excessive phosphate was supplied, TsSP4 should participate in glucan elongation during starch biosynthesis. These results indicated that the enhanced starch production in the phosphorus-replete cultures during the late phase of nitrogen starvation (24−48 h) could be ascribed to the improved SP activities. Other Enzymes Related to Starch Metabolism. To get a more comprehensive picture of the effect of excessive phosphate on starch metabolism, starch synthase (SS), branching enzyme (BE), and isoamylase (ISA) for starch biosynthesis and amylase (AMY) for starch degradation were also analyzed by zymograms. As shown in Figure 6a, only one weak band representing the TsSS activity was detected in the culture with 9 mM phosphorus at 6 and 48 h. SS catalyzes the subsequent step of AGPase catalyzed one, i.e., transferring of the glucosyl from ADP-glucose (ADP-Glc) to a nonreducing end of a growing α-1,4-linked glucan.10 The weak activity of SS along with the declined AGPase activity (Figure 5a) during nitrogen starvation further suggested that AGPase-based starch biosynthesis pathway should not make substantial contributions. As for BE and ISA, the blue band in Figure 6b was assigned as ISA activity due to the formation of linear glucan from amylopectin catalyzed under ISA. The brown band in Figure 6b was assigned as BE activity due to the formation of more branched glucan from amylopectin catalyzed under BE. Two active bands for each enzyme, TsBE1/2 and TsISA1/2, respectively, were detected (Figure 6b). It was evident that phosphorus supply (3 mM and 9 mM) enhanced the activities of both TsBEs and TsISAs, especially at 6 and 24 h. BE catalyzes the formation of branched (α-1,6-linked) glucan chain, while ISA trims and modifies the structure of branched glucan by hydrolyzing the α-1,6-glycosidic bond, both of which play important roles in the biosynthesis of amylopectin.10 The positive correlation of activities of BEs or ISAs with SPs (Figure 6b and Figure 5b) suggested that these enzymes could
Figure 6. Starch synthase (SS), branching enzyme (BE), isoamylase (ISA), and amylase (AMY) activities revealed by zymograms under nitrogen starvation in Tetraselmis subcordiformis under different phosphorus availability conditions. The blue band in part b was assigned as ISA activity due to the formation of linear glucan from amylopectin catalyzed under ISA. The brown band in part b was assigned as BE activity due to the formation of more branched glucan from amylopectin catalyzed under BE.
act coordinately. It has been demonstrated that plastidial SP can form protein complexes with BEs for the coordinate elongation of MOSs during amylopectin biosynthesis in cereals.43 SP could also recycle the MOSs released from the trimming process catalyzed by ISAs.11 Overall, phosphorus could promote starch biosynthesis by enhancing SP performances along with the related MOS-modifying enzymes such as BEs and ISAs in T. subcordiformis. For starch degradation enzyme, two major AMY activities, TsAMY1 and TsAMY2, were detected (Figure 6c). During the early phase within 24 h, the AMY activities declined to undetectable levels in all the cultures, which could reasonably account for the net starch accumulation. Interestingly, during the late phase until 48 h, the AMY activities were induced significantly only in the culture with the highest phosphorus supply (9 mM). It seemed that starch degradation could be strengthened with the addition of high phosphate up to 9 mM. However, the net starch accumulation was in fact enhanced in the 9 mM culture compared with the 0 mM counterpart, and it was also comparable to that in the 3 mM one (Figure 1d). These results indicated that it was the enhancement of starch biosynthesis rather than the abatement of starch degradation 10783
DOI: 10.1021/acs.jafc.8b04798 J. Agric. Food Chem. 2018, 66, 10777−10787
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Journal of Agricultural and Food Chemistry
Figure 7. Analysis of metabolites involved in starch biosynthesis and glycolysis in the algal cells cultivated under phosphorus-deprived (0 mM) and replete (3 mM) conditions in the context of nitrogen starvation (means ± SD, n = 3). The charts with blue bars represented absolute content (ng/ mg DW) of the metabolite tested, while the gray ones represented the peak area (A) of the metabolite detected on the HPLC−MS/MS system per unit mass of algal cells (A/mg DW). The significant increased metabolites (p < 0.1) were marked in red.
enhanced by more than 6-fold in the culture with 3 mM phosphorus compared with the 0 mM counterpart. The positive correlation between starch accumulation and PEP content was also observed in higher plants like potato tubers.46 PEP has been demonstrated to be an activator of AGPase in Chlamydomonas reinhardtii47 and higher plants,48 and the activation effect was especially pronounced under the inhibition state of AGPase caused by phosphate.49 Therefore, the enhanced PEP level was expected to facilitate starch biosynthesis by activating AGPase or alleviating the inhibition effect exerted by a high level of phosphate in the culture with 3 mM phosphorus. Another evident difference in the culture with 3 mM phosphorus relative to the 0 mM counterpart was the improvement of Tre biosynthesis, with the content of the intermediate, Tre-6-P, and Tre increased by 2.4-fold and 85%, respectively (Figure 7, p < 0.1). Tre-6-P has been deemed to be a signaling molecule in plants which regulates plant growth and development by controlling carbohydrate metabolism.50,51 It can induce starch biosynthesis via thioredoxin-mediated activation of AGPase.52 Therefore, the enhancement of Tre-6P could lead to improved starch accumulation in the phosphorus-replete (3 mM) culture. Meanwhile, Tre-6-P was also reported to boost photosynthetic capacity, which resulted in increased cell division and cell wall biosynthesis.53 This might account for the enhanced cell growth and biomass production in the culture with replete phosphorus (3 mM, Figure 1a−c). The present study demonstrated that starch production could be improved by excessive phosphorus supply under nitrogen starvation in T. subcordiformis. The high extracellular
that led to the higher net starch accumulation in response to excessive phosphorus supply. From the zymogram analysis of the starch synthetic enzymes discussed above, it was speculated that SPs should most likely contribute to the improvement of starch biosynthesis. These findings further reflected the importance of SP in the nitrogen starvation-induced storage starch biosynthesis under excessive phosphorus availability conditions in T. subcordiformis. In Chlamydomonas reinhardtii, the plastidial SP, PhoB, has been demonstrated to be indispensable for normal storage starch accumulation.11 The plastidial SP in plants (Pho1) has recently been documented to play positive roles in starch biosynthesis in sink tissues such as rice44 and potato tubers.45 Collectively, it could be deduced that SP should act as an important participator in storage starch accumulation in both microalgae and plants. Thanks to the participation of SP, the starch biosynthesis could not be impeded, or even be reinforced herein, by high phosphorus availability. Metabolite Levels. To further reveal the possible reasons for the positive effect of phosphorus on storage starch accumulation in T. subcordiformis, some metabolites involved in starch biosynthesis and central carbon metabolism were determined in the cultures under phosphorus-deprivation (0 mM) and phosphorus-repletion (3 mM) conditions at 24 h. Unexpectedly, the majority of the metabolites tested showed no significant difference between the two cultures (Figure 7 and Figure S3), except for phosphoenolpyruvate (PEP), trehalose-6-phosphate (Tre-6-P), and trehalose (Tre), all of which displayed increased contents. The most dramatic increase occurred in PEP (p < 0.01), the content of which 10784
DOI: 10.1021/acs.jafc.8b04798 J. Agric. Food Chem. 2018, 66, 10777−10787
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Journal of Agricultural and Food Chemistry
Table 1. Comparison of Biomass/Starch Production Ability and Phosphorus Removal Capacity of Different Microalgae Cultivated under Photoautotrophic (P), Heterotrophic (H), or Mixotrophic (M) Conditions Reported in the Literature species
trophic type
nutritional condition
biomass productivity (g/L/d)
starch content (% DW)
starch productivity (g/L/d)
phosphorus removal rate (mg/L/d)
4
0.035 0.052 0.057 0.18 0.20 0.24 0.45 0.70 0.95 0.325
25.6 26.2 28.9 36 31 28 -a -a -a -a
0.088 0.133 0.158 0.09 0.09 0.10 -a -a -a -a
-a 0.27 0.78 2.33 10.85 9.96 1.375 9.5 13 51.11
cultivation time (d)
Scenedesmus obliquus
P
Chlorella sp.
P
Chlorella regularis var. minima
H
Chlorella kessleri
M
− N − P (0 mM) − N + P (0.21 mM) − N + P (1.45 mM) − N + P (0.17 mM) − N + P (5 mM) − N + P (10 mM) − N + P (0.17 mM) − N + P (1.23 mM) − N + P (1.68 mM) + N (9.3 mM) + P (7.6 mM)
Scenedesmus sp. UM284 Tetraselmis subcordiformis
M
+ N (9.6 mM) + P (6.8 mM)
4
0.248
-a
-a
39.22
P
− N − P (0 mM) −N + P (3 mM) −N + P (9 mM)
2
0.41 0.58 0.56
49.6 64.5 62.6
0.31 0.50 0.48
-a 23.6 45.4
16
2
4
ref Chu et al.18 Zhu et al.16 Fu et al.54 Li et al.55 Zhou et al.56 this study
a
No data available.
phosphorus availability up to 9 mM did not affect biomass and starch production. Furthermore, it was noteworthy that the phosphorus removal rate in T. subcordiformis at 48 h reached 23.6 mg/L/d to 45.4 mg/L/d, which were remarkably higher than most of the microalgae cultivated photoautotrophically (Table 1). The phosphorus removal rate of T. subcordiformis was even comparable to some species (e.g., Scenedesmus sp. UM284) cultivated mixotrophically with sufficient nitrogen supply.56 Since nitrogen availability could facilitate phosphorus assimilation,57 it can be expected that the phosphorus removal rate would be even higher if nitrogen source was supplied. Moreover, the biomass productivity in T. subcordiformis appeared competitive among photoautotrophic microalgae as well as even mixotrophic ones (Table 1), indicating that T. subcordiformis was also a good candidate for photosynthetic CO 2 mitigation. The starch production ability in T. subcordiformis was superior to most of the microalgae reported hitherto, as demonstrated in the previous19,37,58 and present study (Table 1). These findings highlighted the potential of using T. subcordiformis for phosphorus reclamation from high strength phosphorus wastewater (e.g., piggery effluent) coupled with CO2 mitigation and starch production. In conclusion, excessive phosphorus supply was conducive to biomass and storage starch production in T. subcordiformis under nitrogen starvation. The starch content of 62% DW and starch productivity of 0.48 g/L/d in the phosphorus-replete cultures (3 mM and 9 mM) at 48 h were 26% and 55% higher, respectively, than those in the phosphorus-deprived one (0 mM). Phosphorus enhanced energy utilization efficiency for biomass and starch production under nitrogen starvation. The proposed model for the enhancement of nitrogen starvationinduced storage starch accumulation under phosphorusexcessive conditions in T. subcordiformis was depicted in Figure 8. High phosphorus availability facilitated starch biosynthesis via maintaining large cell volumes, enhancing activities of SPs along with the related MOS-modifying enzymes such as BEs and ISAs and increasing PEP and Tre6-P levels to alleviate the inhibition of high phosphate availability to AGPase, all of which improved starch production. T. subcordiformis could be a potential candidate
Figure 8. Proposed model for the enhancement of nitrogen starvation-induced storage starch accumulation under phosphorusexcessive conditions in T. subcordiformis. The enhanced enzyme activities or metabolites were marked in red, while the declined enzyme activity was marked in blue. The red arrow represented an activation effect.
for phosphorus reclamation coupled with CO2 mitigation and starch production.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b04798. Figure S1, semiquantitative analysis of starch content with iodine staining using ImageJ; Figure S2, protein degradation in Tetraselmis subcordiformis under different phosphorus availability conditions; Figure S3, analysis of 10785
DOI: 10.1021/acs.jafc.8b04798 J. Agric. Food Chem. 2018, 66, 10777−10787
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metabolites involved in TCA cycle in the algal cells cultivated under phosphorus-deprived and replete conditions in the context of nitrogen starvation (PDF)
AUTHOR INFORMATION
Corresponding Authors
*Phone/fax: +86 28 85405221. E-mail: yaochanghong@scu. edu.cn. *Phone/fax: +86 411 84379069. E-mail:
[email protected]. ORCID
Changhong Yao: 0000-0003-2527-7391 Song Xue: 0000-0002-4039-7685 Yongkui Zhang: 0000-0003-2478-9758 Funding
This work was supported by the National Natural Science Foundation of China (Grants 41406177 and 31500294) and the Fundamental Research Funds for the Central Universities (Grant YJ201734). Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED RuBP, ribulose-1,5-bisphosphate; 3-PGA, 3-phosphoglycerate; 2-PGA, 2-phosphoglycerate; 1,3-PGA, 1,3-bisphosphoglycerate; GAP, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; Fru-1,6-P, fructose-1,6-bisphosphate; Fru-6-P, fructose-6-phosphate; Glc-6-P, glucose-6-phosphate; Glc-1-P, glucose-1-phosphate; ADP-Glc, ADP-glucose; UDP-Glc, UDPglucose; Tre-6-P, trehalose-6-phosphate; Tre, trehalose; PEP, phosphoenolpyruvate; Pry, pyruvate; AcCoA, acetyl CoA; AGPase, ADP-glucose pyrophosphorylase; SP, starch phosphorylase; SS, starch synthase; BE, branching enzyme; ISA, isoamylase; AMY, amylase.
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