Fu et al.

Biotechnol Biofuels (2019) 12:121

https://doi.org/10.1186/s13068-019-1458-z Biotechnology for Biofuels

RESEARCH Open Access

Hormesis effects of phosphorus

on the viability of Chlorella regularis cells
under nitrogen limitation
Liang Fu1, Qingcheng Li1, Ge Yan1, Dandan Zhou1* and John C. Crittenden1,2

Abstract
Background: Phosphorus (P) is an essential element of microalgae, which is either required for anabolism or for
energy metabolism. When employing a nitrogen limitation strategy to trigger microalgal intracellular lipid accumula-
tion, P supplementation was always simultaneously applied to compensate for the accompanied growth inhibition.
Results: This study identified that P exerts hormesis effects on microalgae. Slight excess of P (≤ 45 mg L−1) under
nitrogen limitation condition stimulated the cell growth of Chlorella regularis and achieved a 10.2% biomass produc-
tion increase. This also improved mitochondrial activity by 25.0% compared to control (P = 5.4 mg L−1). The lipid
productivity reached 354.38 mg (L d)−1, which increased by 39.3% compared to control. Such an improvement was
caused by the intracellularly stored polyphosphate energy pool. However, large excess of P (250 mg L−1) inhibited
the cell growth by 38.8% and mitochondrial activity decreased by 71.3%. C. regularis cells showed obvious poisoning
status, such as enlarged size, plasmolysis, deformation of cell walls, and disorganization of organelles. This is probably
because the over-accumulated P protonated the amide-N and disrupted membrane permeability.
Conclusions: These results provide new insight into the roles of P in microalgae lipid production: P does not always
play a positive role under nitrogen limitation conditions.
Keywords: Microalgae, Phosphorus, Hormesis, Nitrogen limitation, Toxic

Background (adenosine triphosphate)-citrate lyase are stimulated for

Microalgae are a promising feedstock for biodiesel [1, 2]. catalyzing acetyl-CoA production [5, 6]. The lipid con-
In their studies on microalgae bioenergy technologies in tent could be enhanced by 10–50% when nitrogen was
the past 40 years, researchers have endeavored to explore limited [4, 7], and even up to two- to three-fold during
strategies for enhancing biomass production and lipid nitrogen starvation [8, 9].
accumulation [3, 4], which taken together, determines Even though nitrogen deprivation/limitation pro-
microalgal lipid production. Nitrogen limitation is the moted intracellular lipid accumulation, it also
most popular approach to increase the microalgal lipid decreased biomass production by as much as 80%, and
content, because it is able to up-regulate the key enzymes caused the failure of final lipid production improve-
on the lipid biosynthesis pathways: malic enzymes ment [10, 11]. Nitrogen deprivation/limitation could
become more activated for catalyzing pyruvate synthesis weaken proteins synthesis, and thus induce the deple-
and NADPH (reduced nicotinamide adenine dinucleo- tion of ADP (adenosine diphosphate) and NADP (nic-
tide phosphate) formation and both acetyl-CoA and ATP otinamide adenine dinucleotide phosphate) and the
dysfunction of cell synthesis [12, 13]. Phosphorus (P)
supplementation offered an effective strategy to resolve
*Correspondence: zhoudandan415@163.com these problems and has thus been employed to enhance
1
Engineering Lab for Water Pollution Control and Resources Recovery,
School of Environment, Northeast Normal University, Changchun 130117, microalgae biomass production under nitrogen limi-
People’s Republic of China tation [14, 15]. Combining nitrogen limitation with P
Full list of author information is available at the end of the article

© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
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and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/
publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Fu et al. Biotechnol Biofuels (2019) 12:121 Page 2 of 9

supplementation could contribute 10–100% increase on Methods

the microalgae biomass production, accompanied by Microalgae strain, cultivation, and protocols
15–50% improvement of lipid accumulation, and finally As a typical microalga, C. regularis var. minima (FACHB-
achieved 50–160% enhancement of lipid productiv- 729) was used in this study, which was purchased from
ity [4, 15, 16]. In a P-repletion condition, P was stored the Freshwater Algae Culture Collection of the Institute
as intracellular polyphosphate (poly-P) by microal- of Hydrobiology in Wuhan, China. Prior to the experi-
gae both in autotrophic and heterotrophic growth [17, ments, C. regularis was purified via the streaking plating
18]. Poly-P, with phosphoanhydride bonds, was rich method and preserved on agar jelly at 4 °C. Before the
in energy (e.g., ATP), which could be utilized for bio- experiment, C. regularis was activated using classic BG11
synthesis when cells were nitrogen deprived [14]. In medium under photoautotrophic condition [24]. As an
addition, poly-P can also be metabolized to form DNA inoculum of the experiment, the activated C. regularis
(deoxyribonucleic acid), RNA (ribonucleic acid), or cells were collected at the logarithmic phase (20 days of
intermediate products in microalgae [5]. Thus, replete phototrophic cultivation) via centrifuging (6000×g for
phosphorus has traditionally been suggested to benefit 5 min at 4 °C), and then washed and re-suspended three
microalgae growth and lipid accumulation [4, 11]. times with 0.9% sterile physiological saline. After the
Excess P is advantageous for microalgal lipid produc- above procedures, C. regularis was inoculated into the
tion; however, it also increases the cost of cultivation. modified BG11 media, which resulted in an initial cell
Agricultural and industrial wastewater are promising density of 1.5 × 107 cell mL−1 for further heterotrophic at
P sources. Piggery wastewater, dairy wastewater, brew- 28 °C, 160 rpm, in the dark (ZWY-240, Zhicheng Shang-
ery wastewater, food processing wastewater, and rub- hai, China). During the experiments, all sampling opera-
ber mill wastewater, are all rich in P [19–22]. The P tions were accomplished in a clean bench (SW-CJ-1FD,
concentrations in these industrial wastewaters range Airtech, Suzhou, China).
widely from 3 to 330 mg L−1. However, previous stud- Real wastewater with high P concentration also has
ies on P supplementation improved microalgal cul- high levels of organics [21, 25]. Accordingly, glucose
tivation, which was limited by the slight excess of P was selected to simulate organic carbon for microal-
(≤ 45 mg L−1). The question remains whether large gae heterotrophic cultivation [11, 26]. In this study,
excess of P can continuously increase the lipid pro- 10 g L−1 glucose (sterilized using sterile 0.22 mm filter)
duction of microalgae. The answer may be negative. was mixed to the BG11 mediums to create heterotrophic
Whether an environmental agent is beneficial or toxic cultivation. Nitrogen limitation was controlled by add-
depends on its dosage, which is referred to as hormesis. ing 300 mg L−1 ­NaNO3. The form of nitrogen was identi-
Hormesis is a biphasic dose–response to an environ- cal to the BG11 medium; however, its content was only
mental agent, i.e., a low dose stimulation has a benefi- 20% of the classic BG11 [4]; therefore, this condition was
cial effect and a high dose has inhibitory or toxic effects named nitrogen limitation (­Nlim). Furthermore, a series
[23]. Based on this, slight excess of P (≤ 45 mg L−1) of phosphorus (­ PO43−–P) levels were proposed to elabo-
could stimulate cell growth [4]; however, the effects of rate the effects of P concentration (mg L−1), 5.4, 25, 45,
large P excess still remain unknown. The results of this 150, and 250 mg L−1. The 5.4 mg-P L−1 was equal to the
study provide new insight for the application of micro- P concentration of the classic BG11 medium; therefore,
algae in P-bearing wastewater treatment. it was used as control protocol ­(NlimP5.4). The others
In this study, the roles of P on Chlorella regularis were called ­NlimP25, ­NlimP45, ­NlimP150, and ­NlimP250
(C. regularis, model strain) were investigated in a wide in the following. Additionally, ­MgSO4, ­NaCO3, ­CaCl2,
concentration range (5.4–250 mg-P L−1) under nitro- citric acid, ammonium ferric citrate, N ­ a2EDTA, ­H3BO3,
gen limiting conditions. The effects of P concentration ­MnCl2, ­ZnSO4, ­CuSO4, ­CoCl2, and ­NaMoO4 were also
on cell growth (which were determined via cell density supplemented as nutrients and trace elements accord-
changes and substrates consumption), and intracellular ing to a previous study [11]. The medium was autoclaved
lipid accumulation (which was related to the intracel- at 121 °C for 30 min, and the final pH was about 7.1
lular contents) were studied. In addition, mitochondrial adjusted with sterile HCl and NaOH solution and no pre-
activity assay, ultrastructure morphology of cells, intra- cipitation appeared in the medium.
cellular-P storage forms, and chemical bond analyses
were conducted, to identify the potential mechanism Growth profiles and nutrient consumptions
underlying P toxicity. Interestingly, hormesis effects of Microalgal growth was evaluated via cell density [10],
P on microalgae cells were confirmed, which specified obtained with an optical microscope (BX53, Olym-
the toxic of high P concentration on microalgae for the pus, Japan) coupled with a hemocytometer. The results
first time. were determined based on the averages of at least three
Fu et al. Biotechnol Biofuels (2019) 12:121 Page 3 of 9

repetitions. The cell growth rate was calculated according

to Eq. (1):
 
Cell growth rate cell (mL d)−1 = (X2 − X1 )/(t2 − t1 )
(1)
where X1 and X2 (cell mL−1) represent the cell density at
times t1 and t2, respectively; t1 and t2 represent the initial
and final point within the linear portion of Fig. 1a.
Organics, phosphorus, and nitrogen consumptions
were of particular concern, which were represented by
the changing of chemical oxygen demand (COD), dis-
solved inorganic nitrogen (DIN), and dissolved inorganic
phosphorus (DIP) in this work. These were determined
via spectrophotometry using a water quality analyzer and
the provided reagent kits (5B-3C V8, Lian Hua Technol-
ogy, China) according to the manufacturer’s instructions.
Prior to measurements, the samples were filtered with
0.45 µm cellulose acetate membranes to remove any sus-
pended residues and biomass.

Intracellular storage products: lipid, protein, and starch

The cells were collected on day 4 via centrifugation at
10,610×g for 10 min at 4 °C (TGL-16M, Cence, China),
and were then freeze-dried (Pilot1-2LD, Boyikang,
China) at − 80 °C. The achieved lyophilized powder was
used for lipid extraction using the chloroform–methanol
(2:1 v/v) reagent method [27]. Therefore, the lipid con-
tent could be calculated based on the gravimetric ratio of
the extract and the powder and the lipid productivity was
determined based on the following equation [28]:
 
Lipid productivity mg (L d)−1
(2)
= (B2 × C2 − B1 × C1 )/(T2 − T1 )
where B1 and B2 represent the biomass (mg L−1) at the
times T1 and T2, respectively; C1 and C2 represent the
lipid content (%) at times T1 and T2, respectively. T1 and
T2 represent the sampling time at initial and day 4.
To measure protein and starch, the cells were pre-
treated via ultrasound at 1500 W, − 4 °C for 30 min to
break the cell-walls. Then, the solution was filtered with
a 0.45 µm cellulose acetate membrane to remove frag-
ments. The filtrate was ready for intracellular protein Fig. 1 C. regularis growth profiles (a), the cell growth rate (b), and DIP
and starch evaluation. A folin-phenol assay kit (Ding- consumptions (c) when P concentrations were 5.4 mg L−1 ­(NlimP5.4),
guo Changsheng Biotechnology, Beijing, China) was 25 mg L−1 ­(NlimP25), 45 mg L−1 ­(NlimP45), 150 mg L−1 ­(NlimP150),
and 250 mg L−1 ­(NlimP250). Error bars represent standard deviations,
employed for protein analysis [29]. For the hydrolysis of
which were obtained based on triplicate measurements
starch, 30% perchloric acid was added to the filtrate and
stirred at 25 °C for 15 min, the extracts were analyzed
with the anthrone method [30].
lium bromide (MTT) assay kit (Beyotime, Beijing, China)
Mitochondrial activity assay according to a previous publication [31]. Briefly, the cells
The mitochondrial activity was determined with the were seeded to a 96-well plate (5 × 105 cells mL−1) and
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazo- were incubated with fresh air ventilated (5% ­CO2), at
Fu et al. Biotechnol Biofuels (2019) 12:121 Page 4 of 9

37 °C. After 48 h, 10 µL MTT (5 mg mL−1) was added to P < 0.05. All statistical analyses were performed using the
the wells for another 4 h of incubation. The absorbance of SPSS software package (IBM SPSS statistic 20.0).
the culture was measured at 570 nm using a microplate
reader (RT-6000, Rayto, China). Results and discussion
Effect of P concentrations on C. regularis growth
Morphology and ultrastructure of cells The cell density of C. regularis was ~ 4.70 × 107 cell mL−1
Microalgal cell morphology was observed via opti- at the stationary stage when P supply was regular,
cal microscopy (BX53, Olympus, Japan) and scanning ­NlimP5.4 (refers to the classic BG11), see Fig. 1a. The cell
electron microscopes (SEM) (XL-30 FE-SEM, FEI Co., density was about 76% of that in NP5.4 [18], indicating
USA). The ultrastructure of the cell was observed with that nitrogen limitation inhibited cell growth. Microalgal
a transmission electron microscope (TEM) equipped growth was stimulated when P was slightly excessive, i.e.,
with an energy dispersive X-ray spectrometer (EDX) the cell density increased by 10.2% for N ­ limP45 (P < 0.05).
(JEM 1200EX, JEOL, Japan). Prior to SEM and TEM A considerable number of previous studies reported that
observation, the cells were pretreated by a series of such a stimulation was caused by storage of excess P,
multiple fixative and dehydrated procedures [32, 33]. under either autotrophic or heterotrophic cultivations [4,
Before TEM–EDX analysis, the dehydrated cells should 14, 39, 40], where nitrogen was either limited or unlim-
be embedded in epoxy resin (Epon812, Shell Chemical, ited [41]. Excessive P induced improvements on supplying
USA) for cutting (Microtome, Leica UCT, Germany) and phospholipid, genetic materials, and energy for cell divi-
ultrathin sections were further stained with uranyl ace- sion [42, 43]. However, further increasing of the P sup-
tate [34]. ply could result in microalgal growth inhibition. The cell
density decreased for ­NlimP150 and decreased as much as
31
P nuclear magnetic resonance spectroscopy (NMR) 38.8% for ­NlimP250, compared to the control (­NlimP5.4)
The P-storage compounds in microalgal cells were (P < 0.05). In addition, the growth rate in Fig. 1b intuitively
extracted via the ice-cold ­HClO4 method [14, 35], and shows that the effects of P depended on its concentration
then 10% D­ 2O was added to provide a field-frequency (P < 0.05). These results indicate that C. regularis growth
lock. The 31P NMR spectra were acquired at 400 MHz was promoted when P was at an appropriate level; how-
with a 5 mm probe using an NMR spectrometer (Vnmrs- ever, a large excess of P had a negative impact on the cells.
300, Varian Co., USA). The acquisition time was 16 h at This is a typical manifestation of hormesis [44, 45].
room temperature. MestReNova software 11 was used to Glucose, nitrogen, and phosphorus uptake profiles
analyze the peaks in the spectra, and the P compounds were represented by COD, DIN, and DIP changing ver-
were identified by their chemical shifts based on the pre- sus time, respectively, see Additional file 1: Fig. S1 and
viously published spectra [35–37]. Fig. 1c. The organic and nutrient consumptions were
consistent with the growth profiles: stimulated by low P
X‑ray photoelectron spectroscopy (XPS)
concentrations (P ≤ 4.49 × 10−7 mg cell mL−1) and slow-
The binding energies of N1s and P2p were analyzed to ing down at high P levels (P ≥ 14.97 × 10−7 mg cell mL−1).
investigate the character of the intracellular compounds It is worth mentioning that P ­(PO43−–P) was completely
via XPS (ESCALAB 250, Thermo, USA). The sample was consumed within 4 days when P was in slight excess
the lyophilized powder of microalgal cells, which was (less than 45 mg L−1); in contrast, a large amount of P
obtained via the above-mentioned freeze-drying method. ­(PO43−–P) remained for both N ­ limP150 and N ­ limP250
The beam source type was Al K Alpha, and it was operat- (Fig. 1c). These results implied that the capability of P
ing at 250 W, a voltage of 15 kV, a current of 15 mA, an storage reached a threshold, above which, P may cause
energy of 1486.71 eV; the sampling spot size was 500 µm microalgal cell damage.
in diameter and an electron takeoff angle of 50° was used.
The binding energy was calibrated with reference to C1s
at 284.8 eV in the spectra analysis with XPS Peak 4.1 soft- Effects of P concentration on lipid productivity
ware [38]. It seemed that P supply affected the carbon flow of
the microalgal glucose metabolism. The lipid content
enhanced by 22.9% for slightly excessive P ­ (NlimP45)
Statistical analysis
(P < 0.05). However, lipid synthesis was significantly
P concentration effects were investigated based on trip-
suppressed when C. regularis cells were inhibited by
licate cultivations, and then the statistically significances
a large excess of P (­NlimP250), whereas the lipid con-
were analyzed using one-way ANOVA method. The
tent decreased by 15.9% in comparison to the con-
mean values were compared using a least significant dif-
trol ­(NlimP5.4) (P < 0.05). For the cell density (Fig. 1a),
ference (LSD) test. Differences were significant when
Fu et al. Biotechnol Biofuels (2019) 12:121 Page 5 of 9

the highest lipid productivity of 354.38 mg (L d)−1 was

achieved for ­NlimP45, which increased by 39.3% in com-
parison to the control (P < 0.05). However, the lipid pro-
ductivity of C. regularis for NlimP250 was only 47.3% of
­NlimP45 and decreased by 34.2% in comparison to con-
trol (P < 0.05). Accordingly, the intracellular protein and
starch contents both positively correlated with P supply,
as shown in Fig. 2a.
It has previously been reported that P enhances both
microalgal lipid synthesis and production. P-replete
favored the enzymes of the up-regulation of the lipid syn-
thesis pathway [40] and could enhance the biomass pro-
duction by activating phosphofructokinase and pyruvate
kinase related glycolysis [46]. Here, for the first time, the Fig. 3 Effect of P concentrations on mitochondrial activity of C.
toxicity of a large excess of P was addressed in microal- regularis under nitrogen limitation, with P 5.4, 45, and 250 mg L−1
gae. Excessive P dosage does not always increase the lipid ­(NlimP5.4, ­NlimP45, and ­NlimP250, respectively). Error bars were
production of microalgae under nitrogen limitation. obtained based on triplicate measurements

Hormesis mechanism ­NlimP45 had a round shape, integrated structure, and

Effect of P concentration on cell viability intact organelles (Fig. 4a–c). All of these characteris-
Mitochondrial activity was used to study the microbial tics were identical to control cells. In contrast, the cells
viability [31, 47], since more than 95% energy is gener- became folded for ­NlimP250, and several cells enlarged,
ated in mitochondria, which are the “powerhouses” of reaching up to two- to three-fold of the size of the con-
the cell [48]. In this work, the trend of mitochondrial trol (Fig. 4d, e). TEM images further showed that the cell
activity again supported P hormesis deduction, as shown walls thinned and even detached. Such damage of the
in Fig. 3. Mitochondrial activity was stimulated and con- plasma membrane and the following plasmolysis caused
sequently increased by 25.0% (P < 0.05), when P was only the described cell surface folding [49]. In addition, sev-
slightly excessive ­ (NlimP45). However, it decreased as eral organelles, such as mitochondria, were disordered
much as 71.3% for N ­ limP250 (P < 0.05), indicating dys- when P was in large excess (Fig. 4f ). These signs indicate
function of mitochondria due to the toxicity of the large targeted P poisoning of microalgal cells.
excess of P.
Intracellular P distribution and the role of polyphosphates
Cell morphology and ultrastructure P distributions in C. regularis cells were different
Cell morphology and ultrastructure further corrobo- between stimulated (­NlimP45) and inhibited (­NlimP250)
rated the hormesis mechanism of P. C. regularis cells of cell growth. P was mainly found in the intracellular

Fig. 2 Effects of P concentrations on C. regularis intracellular components. Lipid, protein, and starch content (a), and lipid productivity (b) on day 4.
­NlimP5.4, ­NlimP25, ­NlimP45, ­NlimP150, and N
­ limP250 represent the protocols using the modified BG11 media with P concentrations of 5.4, 45, 150, and
250 mg L−1, respectively. Error bars were obtained based on triplicate measurements
Fu et al. Biotechnol Biofuels (2019) 12:121 Page 6 of 9

Fig. 4 Morphology of surface and inner C. regularis cells cultivated under two conditions (nitrogen limitation and P 45 mg L−1, ­NlimP45, a–c;
nitrogen limitation and P 250 mg L−1, ­NlimP250, d–f) with beneficial and inhibition effects for growth. The subfigures in b and e show representative
enlarged views of cells. The red, blue, orange, and green arrows indicate light colored cells, large sized cells, fold structure, and lysed cells devoid of
recognizable contents, respectively. cw cell wall, pm plasma membrane, m mitochondria

region for ­NlimP45 (4.13% in weight), while it was located death at the end [51]. Moreover, excess poly-P was able to
at the cell periphery for ­NlimP250 (5.4% in weight), see form dinucleoside polyphosphates with similar structure
Fig. 5. P in C. regularis formed poly-Ps (small black spots to ATP and other essential mononucleotides; therefore, it
in TEM) in the cells for N ­ limP45, which were at the reso- was also toxic for cells and caused DNA damage in the
nances in − 5 to − 7 and − 17 to − 22 ppm [35, 36] in the intra-S phase [52].
31
P NMR spectra (Fig. 5). The poly-Ps also convinced by Excess poly-P could bind to intracellular components
the peaks at 129.4 eV and 137.1 eV [50] in P 2p XPS spec- and inhibit cell viability [18]. There were several new
tra (Fig. 5). Poly-P served as an energy pool to stimulate peaks at − 7.7 ppm and − 19.8 ppm in the 31P NMR spec-
cell division and metabolism [37]. tra, which indicated that the new form of P appeared for
However, the forms of poly-P became different when ­NlimP250 (Fig. 5). This was also confirmed by the peak at
the excess poly-P was stored for C. regularis ­(NlimP250). higher binding energy of 139.8 eV in the P 2p XPS spec-
The poly-Ps located near the cell periphery, which likely tra (Fig. 5), which represented the binding compounds of
damaged both plasma membrane and cell wall (Fig. 5). poly-P to intracellular components [18]. Furthermore, a
In addition, mitochondria were disordered, as the peaks new peak of protonated amide-N in protein at 402.3 eV
(− 11.5 ppm) of ATP and NADH [35] almost disappeared [53, 54] appeared for N ­ limP250 (Fig. 6). It seems that
in 31P NMR spectra for N ­ limP250 (Fig. 5). This indicated the excess poly-P bonded to the proteins and resulted
that excess poly-Ps damaged the energy production pro- protonated amide-N, which affected the binding of the
cess, which was consistent with the results on growth protein–ligand [55]. Thus, a major constraint in pro-
inhibition and mitochondrial activity decrement. Conse- tein breakage and large cytoplasmic crevices appeared,
quently, the excess poly-P accumulation disintegrated the followed by interfered interactions of protein subunits
thylakoid membranes and resulted the cell lysis and cell [55]. The changes on channel and transporter proteins
Fu et al. Biotechnol Biofuels (2019) 12:121 Page 7 of 9

Fig. 5 31P NMR spectra and P 2p XPS spectra in C. regularis cells, with magnified TEM and P elemental mapping images inserted. The yellow arrows
indicate poly-P granules. The red circle shows the inner cell area, the orange circle shows the cell wall area. The data from the red and orange circles
indicates the weight percentage of P in the circle area, ND means not detected

Conclusions
P presented a hormesis effects for microalgal cultiva-
tion. Slightly excessive P levels (≤ 45 mg L−1) stimulated
C. regularis enhancement of growth (10.2%), lipid accu-
mulation (22.9%), and mitochondrial activity (25.0%)
via poly-P storage energy. The total lipid productiv-
ity increased by 39.3%. In contrast, large excess of P
(P ≥ 150 mg/L) poisoned C. regularis cells, which showed
enlarged size, plasmolysis, deformation of cell walls,
and disorganization of organelles. Both cell density and
mitochondrial activity decreased by 38.8% and 71.3%,
respectively, followed by a decrease of the final lipid pro-
ductivity of 34.2%. The poisoning mechanisms are related
to intracellular amide-N in protein protonation and the
damage of the plasma membrane.

Additional file
Fig. 6 N 1s XPS spectra of C. regularis cells. The microalgal cells were
Additional file 1: Fig. S1. The COD and DIN consumptions during C. regu-
freeze-dried to obtain lyophilized powder for XPS analysis
laris growth with different P concentrations. Error bars represent standard
deviation values, which were obtained based on triplicate measurements.

induced the across membrane process of ions and disor- Abbreviations

dered small molecules [56]. In this way, membrane per- P: phosphorus; NADPH: reduced nicotinamide adenine dinucleotide phos-
meability was damaged and membrane proteins were phate; NADP: nicotinamide adenine dinucleotide phosphate; ATP: adenosine
triphosphate; ADP: adenosine diphosphate; poly-P: polyphosphate; DNA:
disrupted [57].
Fu et al. Biotechnol Biofuels (2019) 12:121 Page 8 of 9

deoxyribonucleic acid; RNA: ribonucleic acid; C. regularis: Chlorella regularis; in the green microalga Dunaliella tertiolecta. J Agric Food Chem.
Nlim: nitrogen limitation; COD: chemical oxygen demand; DIN: dissolved 2017;65:3190–7.
inorganic nitrogen; DIP: dissolved inorganic phosphorus; MTT: 3-(4,5-dimethyl- 7. Griffiths MJ, van Hille RP, Harrison STL. The effect of nitrogen limitation on
2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; SEM: scanning electron lipid productivity and cell composition in Chlorella vulgaris. Appl Micro-
microscopes; TEM: transmission electron microscope; EDX: energy dispersive biol Biotechnol. 2014;98:2345–56.
X-ray spectrometer; NMR: nuclear magnetic resonance; XPS: X-ray photoelec- 8. Li Y, Horsman M, Wang B, Wu N, Lan CQ. Effects of nitrogen sources on
tron spectroscopy. cell growth and lipid accumulation of green alga Neochloris oleoabun-
dans. Appl Microbiol Biotechnol. 2008;81:629–36.
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DZ conceived of this study, LF analyzed the experiment data and drafted the Impacts of nitrogen and phosphorus starvation on the physiology of
manuscript, LF and DZ revised the manuscript, QL and GY carried out micro- Chlamydomonas reinhardtii. J Appl Phycol. 2016;28:1509–20.
algal cultivation and collected the experiment data, QL performed statistical 10. Zhu S, Huang W, Xu J, Wang Z, Xu J, Yuan Z. Metabolic changes of starch
analysis, JCC provided useful suggestions for this study and manuscript. All and lipid triggered by nitrogen starvation in the microalga Chlorella
authors read and approved the final manuscript. zofingiensis. Bioresour Technol. 2014;152:292–8.
11. Shen XF, Chu FF, Lam PKS, Zeng RJ. Biosynthesis of high yield fatty acids
Funding from Chlorella vulgaris NIES-227 under nitrogen starvation stress during
This research was supported by the National Natural Science Foundation of heterotrophic cultivation. Water Res. 2015;81:294–300.
China (51708095, 51578117, 51722803), Fundamental Research Funds for the 12. Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, Darzins
Central Universities (2412017QD027, 2412018ZD013, 2412018ZD042), the A. Microalgal triacylglycerols as feedstocks for biofuel production: per-
Science and Technology Project of Jilin Province (20180520168JH), China Post- spectives and advances. Plant J. 2008;54:621–39.
doctoral Science Foundation (2017M611302, 2018T110241) for their financial 13. Fields MW, Hise A, Lohman EJ, Bell T, Gardner RD, Corredor L, Moll K,
support. We also thank Dr. Yanhong Xiao and Dr. Nan Lu, Experiment Center Peyton BM, Characklis GW, Gerlach R. Sources and resources: importance
of School of Environment, Northeast Normal University for assistance with our of nutrients, resource allocation, and ecology in microalgal cultivation for
experimental data acquisition. lipid accumulation. Appl Microbiol Biotechnol. 2014;98:4805–16.
14. Chu FF, Chu PN, Cai PJ, Li WW, Lam PKS, Zeng RJ. Phosphorus plays an
Availability of data and materials important role in enhancing biodiesel productivity of Chlorella vulgaris
Not applicable. under nitrogen deficiency. Bioresour Technol. 2013;134:341–6.
15. Shen XF, Liu JJ, Chu FF, Lam PKS, Zeng RJ. Enhancement of FAME produc-
Ethics approval and consent to participate tivity of Scenedesmus obliquus by combining nitrogen deficiency with
Not applicable. sufficient phosphorus supply in heterotrophic cultivation. Appl Energy.
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