Bioresource Technology 270 (2018) 588–595

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Production of primary metabolites in Microcystis aeruginosa in regulation of T

nitrogen limitation
⁎
Zhaojiang Zuoa, , Binbin Nia, Lin Yangb
a
School of Forestry and Biotechnology, Zhejiang A & F University, Hangzhou 311300, China
b
Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, Tianjin, 300387, China

A R T I C LE I N FO A B S T R A C T

Keywords: The aim of this work was to study the regulatory effect of nitrogen (N) deficiency on primary metabolites in
Biofuel Microcystis aeruginosa, and promote the utilization of the alga. Low-N and Non-N conditions, especially Non-N,
Microcystis aeruginosa reduced the cell growth and photosynthetic abilities compared to Normal-N, as N deficiency triggered the down-
Nitrogen deficiency regulation of genes involving in the photosynthetic process. Non-N not changed lipid content, due to no up-
Primary metabolite
regulation of genes that promoted lipid synthesis. Soluble protein content significantly decreased under Non-N,
Transcriptome
which may result from the declined expression of genes relating to amino acid and histidyl-transfer RNA
synthesis. Soluble and insoluble carbohydrate content significantly increased under Non-N, as the expression
variation of genes blocked sugar degradation and promoted lipopolysaccharide synthesis. Therefore, M. aeru-
ginosa can be used as the feedstock to produce carbohydrates under N deficiency for bioethanol production, and
the remainder lipids after carbohydrate extraction can be used to produce biodiesel.

1. Introduction widely explored strategy, which can cause carbohydrate accumulation

in Chlorella vulgaris (Dragone et al., 2011; de Farias Silva and Sforza,
Fossil fuels are limited non-renewable resources, and account for 2016), Scenedesmus obliquus (Ho et al., 2012) and Isochrysis zhangjian-
about 85% of the global energy consumption. Their over-consumption gensis (Wang et al., 2014), lipid accumulation in Chlamydomonas re-
will lead to a series of issues in the incoming decades, such as energy inhardtii (Li et al., 2012), Dunaliella parva (Shang et al., 2016a) and
crisis, raise in oil price, depletion of natural resources and climate Phaeodactylum tricornutum (Burch and Franz, 2016; Longworth et al.,
change (Daroch et al., 2013). To address these issues, sustainable bio- 2016), and both accumulation in Neochloris oleoabundans (Morales-
fuels have attracted much attention, and microalgae are considered as Sánchez et al., 2014; Sun et al., 2014) and C. zofingiensis (Zhu et al.,
the promising alternative feedstocks for the biofuel production, due to 2015).
their great photosynthetic efficiency, fast growth rate, high carbohy- N deficiency can improve carbohydrate and/or lipid content in
drate and neutral lipid yields and other numerous environmental ad- microalgal cells, but is not beneficial to the cell growth and biomass
vantages relative to terrestrial plants (Ho et al., 2012). increase (de Farias Silva and Sforza, 2016). Then, a two-stage cultiva-
In microalgae, carbohydrates and neutral lipids are their primary tion strategy has been proposed, which involves growth under N suf-
carbon and energy reserves, which can be used to produce bioethanol ficient condition to high biomass levels then shifting to N deficiency for
and biodiesel (Ho et al., 2012), respectively. Lots of studies have fo- carbohydrate and/or lipid accumulation, e.g., this strategy improved
cused on increasing the accumulation of the 2 kinds of compounds in starch content in C. vulgaris (Dragone et al., 2011), lipid content in
microalgal cells to lower the production cost (Wang et al., 2014; de Chlorella sp. (Zhu et al., 2018) and Tetraselmis marina (Moussa et al.,
Farias Silva and Sforza, 2016; Shang et al., 2017). The accumulation of 2017), and both content in N. oleoabundans (Sun et al., 2014), in-
carbohydrates and lipids can protect microalgal cells to tolerate en- dicating that N deficiency is a signal in triggering the accumulation.
vironmental stresses, so several stressful conditions has been detected Although many studies focused on the accumulation of carbohy-
to improve their content, including element (nitrogen (N), phosphorus drates and lipids in different microalgae, limited reports uncovered the
(P) or sulfur (S)) limitation (Moussa et al., 2017), high light intensity regulating mechanism of N deficiency. ADP-glucose pyrophosphorylase
(Ho et al., 2012), H2O2 (Burch and Franz, 2016) and salinity (Yao et al., and starch phosphorylase were involved in starch synthesis, whose
2013). Among them, N deficiency is perhaps the most effective and encoded genes improved expression in C. vulgaris var L3 under N

⁎
Corresponding author.
E-mail address: [email protected] (Z. Zuo).

https://doi.org/10.1016/j.biortech.2018.09.079
Received 16 August 2018; Received in revised form 14 September 2018; Accepted 15 September 2018
Available online 16 September 2018
0960-8524/ © 2018 Elsevier Ltd. All rights reserved.
Z. Zuo et al. Bioresource Technology 270 (2018) 588–595

starvation (Ikaran et al., 2015). In C. reinhardtii, galactoglycerolipid 2.4. Analysis of Chl fluorescence
lipase gene played an important regulatory role in triacylglycerol (TAG)
accumulation in response to N deprivation (Li et al., 2012), while acyl- Following our previous method (Xu et al., 2017), a certain M. aer-
CoA: diacylglycerol acyltransferase gene served as the regulatory uginosa cultures about 1 × 107 cells were harvested by centrifugation,
function in TAG synthesis in C. vulgaris var L3 (Ikaran et al., 2015). resuspended in the same culture medium of 10 μL, pipetted on a piece
Meanwhile, the transcriptome and proteome were analyzed in C. re- of filter paper, and incubated in darkness for 15 min. Their Chl fluor-
inhardtii (Schmollinger et al., 2014), P. tricornutum (Yang et al., 2013; escence was measured by using a non-modulation Chl fluorescence
Longworth et al., 2016), C. vulgaris (Liu et al., 2016) and D. parva analyzer (YZQ-500, YZQ Technology Co., Beijing, China). The Chl
(Shang et al., 2016a, 2017) under N deprivation. These results provided fluorescence parameter maximum quantum yield of photosystem II
much-valued help in studying the regulatory mechanism of carbohy- (PSII) photochemistry (Fv/Fm) was evaluated using the equation of Fv/
drate and lipid synthesis under N limitation. Fm = Fm − Fo/Fm.
Cyanobacteria are prokaryotic microalgae, whose blooms impact
drinking water supplies, poison aquatic organism, and cause a series of 2.5. Determination of carbohydrate content
environmental and ecological problems, due to the production of algal
toxins and volatile organic compounds (Zuo et al., 2018a,b). It has been The harvested M. aeruginosa cells were dried by using a freezer dryer
reported that cyanobacteria produced simple lipids and fatty acids (LGJ-10C, Four-Ring Science Instrument Plant, Beijing, China). The
which can be easily transesterified into biodiesel (Murata and Nishida, dried alga of 50 mg was ground with a mortar, added in 5 mL distilled
1987). Microcystis aeruginosa is a typical species for cyanobacteria water to dissolve the soluble carbohydrates at 40 °C for 30 min. After
blooms, and widely exists in eutrophic waters. Nitrogen-limited culti- centrifugation at 4000 rpm for 10 min, the supernatant and sediment
vation efficiently promoted carbohydrate accumulation in the cells were used to determine the soluble carbohydrate and insoluble carbo-
(Huang et al., 2018). Palmitic and lauric acids were the main compo- hydrate content, respectively. They were added in a certain distilled
nents of the saturated fatty acids in M. aeruginosa, while oleic and li- water to 15 mL, and then added in 6 M HCl to decompose the poly-
noleic acids were the main components of the unsaturated fatty acids saccharides to monosaccharides in boiling water bath for 1 h. Then the
(Da Rós et al., 2012). After transesterification, the biodiesel produced solution was neutralized with NaOH, and set to 100 mL with addition of
from the microalgal lipids was within the limits of American ASTM distilled water. After centrifugation at 12,000 rpm, 1 mL supernatant
D6751 (Ashokkumar et al., 2014). These results suggest that M. aeru- was added into 1 mL 6% phenol and 5 mL H2SO4, and determined the
ginosa has potential as the feedstock for biofuel production. To promote absorbance at 490 nm. The carbohydrate content (%) was calculated
the development and utilization of M. aeruginosa, we investigated the according to the standard curve of glucose (Zhang et al., 2014).
growth, photosynthetic abilities, and the content of carbohydrates, li-
pids as well as proteins in the cells under different N levels, and ana- 2.6. Measurement of protein content
lyzed the transcriptome to uncover the response mechanism to N de-
ficiency. M. aeruginosa cells centrifuged from 25 mL cultures were re-
suspended with 1 mL of 50 mM buffer solution (4.6 mM KH2PO4 and
2. Material and methods 45.4 mM K2HPO4), crushed with liquid N2, and centrifuged at
12,000 rpm for 25 min at 4 °C. The supernatant was used to determine
2.1. Cell cultures the soluble protein content using coomassie brilliant blue G-250
method described by Bradford (1976).
M. aeruginosa FACHB-912 was cultured in liquid BG11 medium with
17.6 mM NaNO3 as N source, and kept at a regime of 16 h light 2.7. Measurement of lipid content
(30 μmol m−2 s−1) and 8 h dark, with temperature at 23 °C. The cell
cultures were used for the experiments when their density reached to The dried M. aeruginosa cells of approximately 70 mg (m0) were
mid-logarithmic phase. The cell density was determined by using a added in 4 mL chloroform: methanol (2:1 v/v) solution and stirred for
25 × 16 hemocytometer, with each value being the means of 6 repeats. 1 h in a shaker. After centrifugation, the lipids were extracted again,
and the deposited algae were dried and weighed (m1). The photo-
2.2. Treatments with N supply synthetic pigment content (m2) in dried M. aeruginosa cells was de-
termined according to the method Section 2.3. The quantification of the
According to our previous method (Zuo et al., 2018a), M. aeruginosa lipids (ML) was evaluated using the equation of ML = (m0 − m1 − m2)/
cells collected by centrifugation were washed twice with BG11–N m0 × 100%, according to the method of Ramluckan et al. (2014).
(BG11 minus nitrogen) medium to remove the residual N. Then, the
cells were transferred into the media with 0 mM, 8.8 mM and 17.6 mM 2.8. RNA extraction and illumina sequencing
NaNO3, respectively, for Non-N, Low-N and Normal-N treatment, with
the density of 1.6 × 107 cells mL−1. The pH of these media was ad- Following our previous method (Zuo et al., 2018a), total RNA in M.
justed to 7.1. There were 4 conical flasks of cell cultures for each aeruginosa cells was extracted using a Total RNA Extraction System
treatment, and each conical flask contained 300 mL of cell suspensions (Takara, Japan), and mRNA was purified and fragmented to about
and was considered as a repeat. The cell density, photosynthetic pig- 200 nt. These fragments were used as templates to synthesize cDNA.
ment content, Chl fluorescence were measured every day for 5 days. After purification and addition of a terminal A at the 3′ ends, they were
The content of carbohydrates, proteins and lipids was determined after ligated with sequencing adaptors and performed polymerase chain re-
1, 3 and 5 days. The transcriptomes in Non-N and Normal-N treatments action (PCR) with addition of Phusion high-fidelity DNA polymerase,
were analyzed after 1 day. universal PCR primers and index (X) primer. After purification, their
sequencing was performed in Novogene Bioinformatics Technology Co.
2.3. Measurement of photosynthetic pigment content (Beijing, China).

Cell pellets were collected from 3 mL M. aeruginosa cultures by 2.9. Sequence annotation
centrifugation at 6000 rpm for 8 min. Their chlorophylls (Chls) and
carotenoids (Car) were extracted using 3 mL 80% acetone, and de- Clean reads obtained from editing raw reads were mapped onto
termined according to our previous method (Zuo et al., 2018b). unigene sequences using Bowtie2-2.2.3. The method of expected

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number of Fragments Per Kilobase of transcript sequence per Millions the Chl fluorescence parameter Fv/Fm is an intrinsic and sensitive in-
base pairs sequenced (FPKM) was performed to quantify gene expres- dicator to stressful conditions (Chen et al., 2018). When I. zhangjian-
sion levels, and DESeq R package was used to analyze the differential gensis (Wang et al., 2014) and C. vulgaris (Ikaran et al., 2015) cells were
expression genes (DEGs) in M. aeruginosa cells between under Non-N depleted N, a remarkable reduction was detected in Fv/Fm. In the
and Normal-N condition with significantly differential expression at P- present study, Fv/Fm in M. aeruginosa cells under Low-N and Non-N
value < 0.05 and fold change ≥1. The Kyoto Encyclopedia of Genes conditions significantly (P < 0.05) reduced in contrast to under
and Genomes (KEGG) pathways for DEGs were annotated by KEGG Normal-N from the 2nd day, but there was no significant difference
automatic annotation server (Zuo et al., 2018a). between the 2 conditions (Fig. 2C). Chls and Car are essential photo-
synthetic pigments in algae. Their content decreased under N limita-
2.10. Calculations and statistical analyses tion, which can cause a reduction in capturing light and transducing it
to biochemical energy, as well as Fv/Fm. Meanwhile, the changes of
The data were analyzed through one-way ANOVA using Origin 8.0 thylakoid membrane supramolecular architecture under N starvation
(Origin Lab, USA), and the graphs were drawn using the same software. (Zhao et al., 2016) might also lead to the decline of Fv/Fm. The de-
clined Fv/Fm suggested that microalgal cells were suffering stress after
3. Results and discussion N limitation, resulting in a reduction in photosynthetic abilities (de
Farias Silva and Sforza, 2016).
3.1. M. aeruginosa cell growth with different N supply
3.3. Variations of carbohydrate, lipid and protein levels
M. aeruginosa cells can grow under Normal-N, Low-N and Non-N
conditions, but the lowest growth rate was under Non-N. After 5 days, Carbohydrates, proteins and lipids are 3 primary metabolites in
the cell density under Low-N and Non-N was 7.4% (P < 0.05) and organisms. For carbohydrates, they are complex mixtures, mainly in-
17.6% (P < 0.05), respectively, lower than that under Normal-N cluding monomers and polymers of sugars and sugar derivatives such as
(Fig. 1). That was consistent with previous studies in Microcystis (Huang amino sugars and uronic acids (Templeton et al., 2012). Polymers vary
et al., 2018), I. zhangjiangensis (Wang et al., 2014), P. tricornutum (Yang widely in molecular weights that depend on the degree of poly-
et al., 2013) and C. vulgaris (Ikaran et al., 2015; de Farias Silva and merization, serve as structural (cellulose) or stored (starch) function,
Sforza, 2016) under N limitation. make up large fraction in biomass sources, and can be used to produce
biofuel by hydrolysis (Templeton et al., 2012). In M. aeruginosa cells,
Non-N condition significantly increased the content of soluble carbo-
3.2. Changes of photosynthetic abilities
hydrates, mainly monosaccharides and some oligosaccharides, with an
increase of 29.1% (P < 0.05) and 68.4% (P < 0.05), respectively,
Under Normal-N condition, the Chl content in M. aeruginosa cells
relative to Normal-N after 3 days and 5 days. Compared to Normal-N
kept relatively stable during the 5-day cultivation, while decreased
condition, both Low-N and Non-N conditions improved the content of
gradually under other two conditions, especially under Non-N (Fig. 2A).
insoluble carbohydrates, mainly structural or stored polysaccharides,
The Car content in Normal-N treatment slightly increased from
from the 3rd day and 1st day, respectively, and the promoting effects
0.48 μg 10−7 cells to 0.53 μg 10−7 cells from the 1st day to 5th day,
reached to the maximum after 5 days, with an increase of 19.2%
kept relatively stable in Low-N treatment, but declined gradually in
(P < 0.05) and 28.5% (P < 0.05), respectively. For total carbohy-
Non-N treatment from the 2nd day (Fig. 2B). Similarly, a gradually
drates, Low-N and Non-N treatments increased their content, with an
decrease was also detected in Chl content in P. tricornutum cells
increase of 16.0% (P < 0.05) and 49.4% (P < 0.05), respectively,
(Longworth et al., 2016) and mixed cyanobacterial cultures (Arias
after 5 days (Table 1). Similarly, carbohydrate content in Microcystis
et al., 2018) with prolonging N limiting time, as well as in Microcystis
gradually increased with reducing N concentration from 100 mg L−1 to
(Huang et al., 2018) and C. vulgaris (Liu et al., 2016) cells with reducing
10 mg L−1 (Huang et al., 2018), and C. vulgaris (Dragone et al., 2011; de
N levels.
Farias Silva and Sforza, 2016), S. obliquus (Ho et al., 2012) and I.
Chl fluorescence can rapidly investigate status of photosynthetic
zhangjiangensis (Wang et al., 2014) cells accumulated more carbohy-
apparatus and PSII photochemical efficiency without invasiveness, and
drates with lowered initial N concentration.
In microalgal cells, proteins account for their content from 7% to
40% (Becker, 2007). Under N deprivation, the protein content in I.
zhangjiangensis (Wang et al., 2014), C. zofingiensis (Zhu et al., 2015) and
N. gaditana (Janssen et al., 2018) remarkably declined compared to N
sufficient condition. When T. marina (Moussa et al., 2017) and 3
Chlorella strains (Ördög et al., 2016) were grown in the medium with
different N concentration, the protein content decreased with reducing
N concentration. Similar results were also detected in M. aeruginosa
cells under Low-N and Non-N conditions, especially Non-N, and the
reduction of protein content aggravated with prolonging the N starva-
tion (Fig. 3A).
N is a major element in the composition of proteins, so their
synthesis is prevented when this nutrient is limited in the medium
(Sturme et al., 2018). It is well accepted that N-rich compounds (pro-
teins and pigments) are broken under N limitation and used to support
time-restricted growth process (Ikaran, et al., 2015). Meanwhile, N
starvation shifted the carbon flux from protein synthesis to carbohy-
drate and/or lipid (energy-storage compounds) synthesis (Zhu et al.,
2015). However, the synthetic products depend on microalgal species,
Fig. 1. Effects of different N supply on M. aeruginosa cell growth. Non-N, Low-N e.g., C. reinhardtii (Li et al., 2012), N. oleoabundans (Morales-Sánchez
and Normal-N: The cells were supplied with 0 mM, 8.8 mM and 17.6 mM et al., 2014), C. zofingiensis (Zhu et al., 2015), C. vulgaris (Liu et al.,
NaNO3, respectively. Means ± SE (n = 4). 2016; Ördög et al., 2016), D. parva (Shang et al., 2016a) and P.

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Fig. 2. Effects of different N supply on chlorophylls (A), carotenoids (B) and Fv/Fm (C) in M. aeruginosa cells. Non-N, Low-N and Normal-N: The cells were supplied
with 0 mM, 8.8 mM and 17.6 mM NaNO3, respectively. Means ± SE (n = 4).

tricornutum (Longworth et al., 2016) synthesized and accumulated li- 3.4. Expression of genes in response to N deficiency
pids in cells, while Microcystis (Huang et al., 2018), S. obliquus (Ho
et al., 2012) and I. zhangjiangensis (Wang et al., 2014) accumulated Under stresses, the expression of genes rapidly changed in response
carbohydrates. In the present study, Low-N and Non-N treatments not to the variation of the condition. To uncover the responses of genes in
significantly affected the lipid content during the 5-day cultivation M. aeruginosa cell metabolism to N deficiency, the transcripts involving
(Fig. 3B), suggesting that N limitation only promoted the partitioning of in the photosynthesis and major metabolic pathways including carbo-
carbon to carbohydrates in the growing cells, and N deprivation may be hydrate, N and lipid metabolism were identified according to KEGG
more beneficial to soluble carbohydrate partitioning than insoluble pathway analysis.
carbohydrate (Table 1). In M. aeruginosa, precorrin-6y C5,15-methyltransferase and pre-
Carbohydrates are the main feedstocks for bioethanol production. corrin-4 C11-methyltransferase were encoded by MAE_02680 and
Under Non-N for 5 days, the total carbohydrate content increased to MAE_25690, respectively, serving as catalyzing function in precorrin
60.5% of M. aeruginosa cell dry weight, while the lipid content about formation in Chl synthesis. Protochlorophyllide reductase catalyzes the
18% not significantly changed (Table 1), demonstrating that the mas- formation of chlorophyllide a from protochlorophyllide with NADPH
sively accumulated carbohydrates can be used to produce bioethanol supplying H, and includes light-dependent and light-independent pro-
while the remainder lipids can also be extracted to produce biodiesel, tochlorophyllide reductases (Cahoon and Timko, 2000). The light-in-
due to the good characteristics of the biodiesel with similarity with dependent protochlorophyllide reductase subunit L was encoded by
palm oil after enzymatic transesterification (Da Rós et al., 2012; MAE_16230 in M. aeruginosa. Compared to Normal-N, the 3 genes
Ashokkumar et al., 2014). down-regulated by 45.3%, 51.9% and 62.2%, respectively, under Non-
N condition (Table 2). N is the most essential element for Chls, and its

Table 1
Effects of different N levels on the carbohydrate content in M. aeruginosa.
Soluble carbohydrate content (%) Insoluble carbohydrate content (%) Total carbohydrate content (%)

1 day 3 day 5 day 1 day 3 day 5 day 1 day 3 day 5 day

Normal-N 23.3 ± 1.6a 22.3 ± 1.1b 21.2 ± 1.1b 16.9 ± 0.5b 16.6 ± 1.4b 19.3 ± 0.6b 40.2 ± 1.4b 38.9 ± 2.1b 40.5 ± 0.6c
Low-N 23.1 ± 2.2a 25.5 ± 4.8ab 24.0 ± 0.4ab 19.4 ± 1.0ab 22.0 ± 0.6a 23.0 ± 1.1a 42.6 ± 2.9ab 47.6 ± 4.2ab 47.0 ± 1.5b
Non-N 23.9 ± 1.7a 28.8 ± 4.0a 35.7 ± 1.2a 21.0 ± 1.5a 22.9 ± 4.2a 24.8 ± 1.7a 44.9 ± 0.6a 51.7 ± 8.2a 60.5 ± 0.5a

In the same column, different lowercase letters indicate the significant difference at P < 0.05. Means ± SE (n = 4).

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Fig. 3. Effects of different N supply on soluble protein (A) and lipid (B) content in M. aeruginosa cells. Non-N, Low-N and Normal-N: The cells were supplied with
0 mM, 8.8 mM and 17.6 mM NaNO3, respectively. Means ± SE (n = 4).

deficiency limited Chl synthesis in M. aeruginosa cells not only for in the regeneration. The down-regulation of the two genes (MAE_30020
lacking feedstock but also as a signal triggering down-regulation of and MAE_14970) demonstrated the decline of RuBP, which may also
genes involving in the synthetic process. Biliverdin reductase catalyzes affect CO2 assimilation (Table 2). These results were consistent with the
the opening of tetrapyrrole, whose encoded gene MAE_61890 down- down-regulation of genes encoding Rubisco in C. vulgaris (Ikaran et al.,
regulated under Non-N (Table 2), indicating that Chl degradation was 2015) and E. oleoabundans (Sturme et al., 2018), and the levels of Ru-
inhibited. That may be a strategy for M. aeruginosa cells to maintain Chl bisco and enzymes involving in RuBP regeneration (Longworth et al.,
levels in adapting N deficiency. 2016; Shang et al., 2017) under N limitation.
In addition to Chls and Car, phycobilins also play important roles in In plants and algae, there are 3 main pathways for sugar metabo-
capturing light energy in cyanobacteria, and usually bound with certain lism, including glycolysis, tricarboxylic acid cycle (TAC) and pentose
proteins to form phycobiliproteins serving as antenna proteins, in- phosphate pathway. Pyruvate is the end-product of glycolysis, which
cluding phycoerythrins, phycocyanins and allophycocyanins (Tomitani comes from the conversation of phosphoenolpyruvate catalyzing by
et al., 1999). In the present study, the expression levels of 9 genes pyruvate kinase. Meanwhile, pyruvate can be transformed to phos-
(MAE_24450, MAE_24460, MAE_24480, MAE_21640, MAE_48340, phoenolpyruvate in the catalysis of phosphoenolpyruvate synthase
MAE_49370, MAE_10240, MAE_10260 and MAE_10270) encoding (Burnell, 2010). The genes (MAE_55440 and MAE_02620) of the two
phycobiliproteins declined in Non-N treatment. In photosynthetic enzymes up-regulated by 24.6% and 36.6%, respectively, in M. aeru-
electron transfer chain, lower expression levels were detected in lots of ginosa under Non-N, demonstrating that the microalgal cells may reg-
genes under Non-N condition, including encoding PSII (MAE_54390, ulate the levels of pyruvate (Table 2). In the catalysis of pyruvate de-
MAE_11820, MAE_43890, MAE_54000 and MAE_44250), PSI hydrogenase, pyruvate is converted into acetyl-CoA that enters into
(MAE_23300, MAE_47290 and MAE_36010), ferredoxin (MAE_36530), TAC and is degraded completely. MAE_59640 encoded the E2 compo-
ferredoxin-NADP oxidoreductase (MAE_12570), and ATP synthase nent of this enzyme in M. aeruginosa, and its down-regulation under
(MAE_00920, MAE_50130, MAE_00930, MAE_50160, MAE_50150, Non-N demonstrated the low level of the enzyme, which may decrease
MAE_50170, MAE_50110, MAE_50140 and MAE_50120) (Table 2). The pyruvate entering into TCA. Malate dehydrogenase serves as catalyzing
down-regulation of the genes involving in phycobiliproteins, photo- function in the conversation from malate to oxaloacetic acid that is the
systems and ferredoxin can reduce the light capture, Fv/Fm (Fig. 2C), as substrate for bonding acetyl-CoA in TCA. The increased expression of
well as electron production and transport in M. aeruginosa, while the the encoded gene (MAE_07860) may benefit the substrate supply and
down-regulation of genes involving in ferredoxin-NADP oxidoreductase TCA proceeding. In addition, phosphate acetyltransferase can catalyze
and ATP synthase can decrease the assimilatory power (NADPH and acetyl phosphate to form acetyl-CoA, whose encoded gene
ATP), indicating that a decline of photosynthetic abilities had occurred (MAE_60960) up-regulation might be beneficial to the TCA (Table 2).
under N deficiency. Similarly, the down-regulation was also detected in The benefits for TCA should be a sufficient usage strategy of sugars to
the genes involving in light harvesting complexes in P. tricornutum adapt N deficiency.
(Yang et al., 2013) and photosystems in E. oleoabundans (Sturme et al., 6-Phosphogluconate dehydrogenase (Adams et al., 1994) and
2018), and in the proteins involving in photosystems and ATP synthase deoxyribose-phosphate aldolase (Hoffee, 1968) are two enzymes in
in P. tricornutum (Longworth et al., 2016) and D. parva (Shang et al., pentose phosphate pathway, which catalyze the formation of ribulose
2017) under N limitation. 5-phosphate from 6-phosphogluconate, and D-glyceraldehyde 3-phos-
In carbon fixation, ribulose-1,5-bisphosphate carboxylase/oxyge- phate from 2-deoxy-D-ribose 5-phosphate, respectively. In M. aerugi-
nase (Rubisco) is the initial enzyme catalyzing ribulose-1,5-bispho- nosa, 6-phosphogluconate dehydrogenase gene (MAE_15220) up-regu-
sphate (RuBP) and CO2 to form 3-phosphoglycerate. This compound is lated under Non-N condition, which promoted the formation of C5
converted into 1,3-diphosphoglycerate in catalysis of phosphoglycerate sugar. However, deoxyribose-phosphate aldolase gene (MAE_61230)
kinase. Then, NAD(P)-dependent glyceraldehyde-3-phosphate dehy- down-regulated under Non-N. Meanwhile, two enzymes in RuBP re-
drogenase catalyzes 1,3-diphosphoglycerate to form the first sugar generation, such as fructose-1,6-bisphosphatase II/ sedoheptulose-1,7-
compound glyceraldehyde-3-phosphate. Rubisco consists of large and bisphosphatase and transketolase, also functioned in the degradation of
small subunits (rbcL and rbcS). Their encoded genes (MAE_47890 and C5 sugar in pentose phosphate pathway. Their encoded genes
MAE_47870), phosphoglycerate kinase gene (MAE_43670) and NAD(P)- (MAE_30020 and MAE_14970) down-regulated under Non-N condition
dependent glyceraldehyde-3-phosphate dehydrogenase gene (Table 2). The declined expression of the 3 genes resulted in the de-
(MAE_25030) in M. aeruginosa declined expression under Non-N con- crease of the corresponding enzymes, which can block the further de-
dition, suggesting that the CO2 assimilation was limited. With CO2 as- gradation of C5 sugar.
similation proceeding, RuBP reduces and needs to be regenerated from In starch synthesis, glucose-1-phosphate adenylyltransferase serves
glyceraldehyde-3-phosphate. Fructose-1,6-bisphosphatase II/sedo- as catalyzing function in the conversation from α-D-glucose 1-phosphate
heptulose-1,7-bisphosphatase and transketolase played important roles to ADP-glucose that is used to synthesize starch. In M. aeruginosa, the

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Table 2
Varied expression of genes relating to photosynthesis and metabolism of carbohydrates, N and lipids in response to N deficiency.
Gene ID Description Normal-N readcount Non-N readcount Fold change Non-N vs.
Normal-N

Photosynthesis MAE_02680 Precorrin-6y C5,15-methyltransferase 121.3 ± 10.7 66.3 ± 3.2 −45.3%

MAE_25690 Precorrin-4 C11-methyltransferase 34.7 ± 10.3 16.7 ± 2.9 −51.9%
MAE_16230 Light-independent protochlorophyllide reductase subunit L 785.3 ± 305.2 296.7 ± 85.6 −62.2%
MAE_61890 Biliverdin reductase 114.3 ± 12.3 60.3 ± 3.8 −47.2%
MAE_24450 Phycocyanin beta subunit 95204.3 ± 8419.0 28971.7 ± 5000.0 −69.6%
MAE_24460 Phycocyanin alpha subunit 65000.3 ± 7549.0 18904.3 ± 4078.1 −70.9%
MAE_24480 Phycobilisome rod linker polypeptide 9478 ± 1777.0 3413.3 ± 492.5 −64.0%
MAE_21640 Phycobilisome core component 1099.0 ± 159.3 578.7 ± 55.2 −47.3%
MAE_48340 Phycobilisome rod-core linker polypeptide 13536.7 ± 952.2 5861.7 ± 672.2 −56.7%
MAE_49370 Phycobilisome core-membrane linker polypeptide 17709.7 ± 2047.4 8951.3 ± 502.7 −49.5%
MAE_10240 Phycobilisome small core linker polypeptide 2551.7 ± 586.8 899.7 ± 220.2 −64.7%
MAE_10260 Allophycocyanin beta subunit 16838.3 ± 4782.5 5289.3 ± 1182.7 −68.6%
MAE_10270 Allophycocyanin alpha subunit 14726.7 ± 4567.7 4276.7 ± 978.9 −71.0%
MAE_54390 Photosystem II 13 kDa protein 101.7 ± 1.2 31.3 ± 4.3 −69.2%
MAE_11820 Photosystem II cytochrome c550 1207.7 ± 153.5 493.3 ± 68.4 −59.1%
MAE_43890 Photosystem II psbT protein 48.0 ± 6.5 24.0 ± 1.0 −50.0%
MAE_54000 Photosystem II reaction center protein H 2219.0 ± 88.2 1298.0 ± 123.6 −41.5%
MAE_44250 Photosystem II oxygen-evolving enhancer protein 1 4887.3 ± 355.6 1398.7 ± 197.1 −71.4%
MAE_23300 Photosystem I subunit II 12257.3 ± 633.2 4363.3 ± 1003.0 −64.4%
MAE_47290 Photosystem I subunit III 14165.3 ± 550.0 4926.0 ± 1039.4 −65.2%
MAE_36010 Photosystem I subunit X 175.0 ± 24.5 73.7 ± 8.8 −57.9%
MAE_36530 Ferredoxin I 8535.0 ± 749.5 4197. 7 ± 917.5 −50.8%
MAE_12570 Ferredoxin-NADP oxidoreductase 7314. 7 ± 409.5 4619.3 ± 460.9 −36.8%
MAE_00920 ATP synthase beta subunit 7772.3 ± 289.0 2744.7 ± 76.8 −64.7%
MAE_50130 ATP synthase subunit b 234. 7 ± 29.0 89.3 ± 9.0 −61.9%
MAE_00930 ATP synthase F1 sector epsilon subunit 1765.0 ± 76.3 716. 7 ± 39.8 −59.4%
MAE_50160 ATP synthase CF1 alpha chain 1549.0 ± 19.6 640.0 ± 43.7 −58.7%
MAE_50150 ATP synthase CF1 delta chain 237.0 ± 6.9 94.3 ± 13.5 −60.2%
MAE_50170 ATP synthase CF1 gamma chain 847.0 ± 13.9 396.3 ± 52.7 −53.2%
MAE_50110 ATP synthase CF0 A chain 3747.0 ± 53.5 1711.7 ± 49.0 −54.3%
MAE_50140 ATP synthase CF0 B chain 172.7 ± 6.9 65.7 ± 5.5 −62.0%
MAE_50120 ATP synthase CF0 C chain 4087.0 ± 121.2 1581.0 ± 107.0 −61.3%
MAE_47890 Ribulose-1,5-bisphosphate carboxylase/oxygenase large 14701.7 ± 2692.6 3344.3 ± 687.6 −77.3%
subunit
MAE_47870 Ribulose bisphosphate carboxylase small subunit 2121.7 ± 230.2 546.3 ± 76.7 −74.2%
MAE_43670 Phosphoglycerate kinase 6566.3 ± 326.5 2630.3 ± 195.4 −59.9%
MAE_25030 NAD(P)-dependent glyceraldehyde-3-phosphate 765.0 ± 88.8 377.0 ± 31.3 −50.7%
dehydrogenase
MAE_30020 Fructose-1,6-bisphosphatase II / sedoheptulose-1,7- 3448.3 ± 293.5 1421.0 ± 76.1 −58.8%
bisphosphatase
MAE_14970 Transketolase 2259.7 ± 292.8 1222.0 ± 126.8 −45.9%

Carbohydrate MAE_55440 Pyruvate kinase 310.0 ± 75.4 386.3 ± 81.3 24.6%

metabolism MAE_02620 Phosphoenolpyruvate synthase 49273.7 ± 5791.1 67316.7 ± 3843.0 36.6%
MAE_59640 Pyruvate dehydrogenase E2 component 235.0 ± 49.5 121.3 ± 14.2 −48.4%
MAE_07860 Malate dehydrogenase 88.0 ± 14.0 118. 7 ± 23.1 34.8%
MAE_60960 Phosphate acetyltransferase 49.7 ± 6.4 93.7 ± 17.9 88.6%
MAE_15220 6-Phosphogluconate dehydrogenase 2730.0 ± 476.8 3574.0 ± 772.1 30.9%
MAE_61230 Deoxyribose-phosphate aldolase 24.0 ± 1.5 10.0 ± 0.6 −58.3%
MAE_30020 Fructose-1,6-bisphosphatase II/sedoheptulose-1,7- 3448.3 ± 293.5 1421.0 ± 76.1 −58.8%
bisphosphatase
MAE_14970 Transketolase 2259.7 ± 292.8 1222.0 ± 126.8 −45.9%
MAE_28050 Glucose-1-phosphate adenylyltransferase 1983.0 ± 301.3 773.7 ± 41.3 −61.0%
MAE_39000 UDP-N-acetylglucosamine acyltransferase 85.3 ± 20.4 134.3 ± 35.8 57.4%

Nitrogen metabolism MAE_09050 Glutamine synthetase 960.3 ± 203.9 3303.7 ± 668.3 244.0%
MAE_29110 Glutamate synthase (ferredoxin) 379.3 ± 82.1 479.7 ± 106.2 26.4%
MAE_57980 Hydroxylamine reductase 2411.3 ± 618.4 1441.0 ± 356.4 −40.2%
MAE_08260 Glutamate dehydrogenase (NADP+) 6548.0 ± 454.4 4163.3 ± 444.4 −36.4%
MAE_09700 Ketol-acid reductoisomerase 624.7 ± 218.7 187.0 ± 17.6 −70.1%
MAE_53130 3-Isopropylmalate dehydratase large subunit 584.3 ± 99.4 353.3 ± 49.2 −39.5%
MAE_61200 ATP phosphoribosyltransferase 29.7 ± 7.7 12.0 ± 3.2 −59.6%
MAE_53430 Arginine biosynthesis bifunctional protein 64.7 ± 5.2 30.3 ± 2.9 −53.1%
MAE_17060 Threonine synthase 3362.3 ± 669.4 5470.3 ± 1123.5 62.7%
MAE_55900 Pyrroline-5-carboxylate reductase 1.0 ± 0.0 5.6 ± 1.8 466.7%
MAE_41680 Histidyl-tRNA synthetase 540.0 ± 69.4 222.7 ± 15.8 −58.8%
MAE_05630 Protein-export membrane protein 347.3 ± 60.4 207.0 ± 21.5 −40.4%
MAE_31960 Preprotein translocase SecA subunit 467.0 ± 125.4 284.0 ± 56.2 −39.2%

Fatty acid metabolism MAE_19480 Glycerol-3-phosphate acyltransferase PlsX 107.7 ± 20.4 62.0 ± 5.1 −42.4%
MAE_55360 Phosphatidate cytidylyltransferase 377.7 ± 49.5 234.0 ± 20.4 −38.0%
MAE_01820 Enoyl-[acyl-carrier-protein] reductase 242.7 ± 32.8 95.0 ± 10.1 −60.9%
MAE_49340 Aldehyde-alcohol dehydrogenase 562.7 ± 60.0 811.0 ± 126.1 44.1%

The significantly differential expression at P-value < 0.05 and fold change ≥1. Minus indicates the down-regulation of genes under Non-N condition compared to
Normal-N. Means ± SD (n = 3).

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encoded gene (MAE_28050) of the enzyme down-regulated, which may 2009). The genes (MAE_19480, MAE_55360 and MAE_01820) of the 3
affect starch synthesis (Table 2). UDP-N-acetylglucosamine acyl- enzymes in M. aeruginosa down-regulated in Non-N treatment.
transferase transfers a R-3-hydroxyacyl chain from R-3-hydroxyacyl MAE_49340 encoded aldehyde-alcohol dehydrogenase that played a
acyl carrier protein to the glucosamine 3-OH group of UDP-N-acetyl-α- catalyzing role in the lipid degradation, and up-regulated under Non-N
D-glucosamine (Williams et al., 2006). The transferred product UDP-3- condition (Table 2). The variation of the 4 genes and no up-regulation
O-(3-hydroxy-tetradecanoyl)-N-acetyl-α-D-glucosamine is used to syn- of genes benefiting lipid synthesis were not beneficial to lipid accu-
thesize lipopolysaccharides. MAE_39000 encoded UDP-N-acet- mulation, which might cause the not increased lipid content in M.
ylglucosamine acyltransferase in M. aeruginosa and up-regulated by aeruginosa cells under N deficiency (Fig. 2B) (Huang et al., 2018).
57.4% under Non-N condition, which can promote the formation of
lipopolysaccharides (Table 2). That may be a protection strategy to N 4. Conclusion
deficiency, as lipopolysaccharides always accumulate around the cell
wall as protective compounds in resisting stressful conditions (Gemma N deficiency reduced M. aeruginosa photosynthetic abilities by
et al., 2016). triggering the down-regulation of genes involving in Chl synthesis,
It can be speculated that the increase of insoluble carbohydrate antenna proteins, photosynthetic electron transfer chain, and carbon
content may be mainly from the accumulation of lipopolysaccharides, fixation, which affected the cell growth. Under Non-N, a decrease was
and that the blockage of pyruvate entering into TCA and C5 sugar de- detected in the protein content, while no significant change in lipid
gradation in pentose phosphate pathway may cause the increase of content. The content of soluble and insoluble carbohydrates sig-
soluble carbohydrate content (Table 1). nificantly increased, as the expression variation of genes blocked sugar
In N metabolism, the NH4+ absorbed and converted by nitrate and degradation and promoted lipopolysaccharide synthesis. Therefore, the
nitrite is transferred to glutamate to form glutamine in the catalysis of accumulated carbohydrates under N deficiency can be used to produce
glutamine synthetase. Then, glutamate synthase transfers a NH4+ from bioethanol, while the remainder lipids after carbohydrate extraction
glutamine to ketoglutaric acid to form 2 molecules of glutamate for cell can also be extracted to produce biodiesel for sufficient usage.
usage, as it is a precursor of many metabolites. Under N limitation, the
up-regulation was detected in the expression of the 2 enzyme genes in Acknowledgements
E. oleoabundans (Sturme et al., 2018), and in the enzyme levels in D.
parva (Shang et al., 2017). Consistently, the 2 genes (MAE_09050 and This research was supported by the Natural Science Foundation of
MAE_29110) also up-regulated in M. aeruginosa in Non-N treatment, Zhejiang Province (No. LY17C160004), the National Natural Science
suggesting that N assimilation can be enhanced to supply the N de- Foundation of China (No. 31300364, 31870585, 31700443), and the
mands under lacking condition (Shang et al., 2017). Meanwhile, the School Development Fund for Scientific Research Personnel Startup
genes (MAE_57980 and MAE_08260) of the enzymes that promoted Project (No. 2013FR069).
NH4+ release from assimilated N compounds down-regulated in Non-N
treatment, which might reduce the loss of limited N element in cells. References
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