Bioresource Technology 285 (2019) 121334

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

Enhancing Haematococcus pluvialis biomass and γ-aminobutyric acid T

accumulation by two-step cultivation and salt supplementation
⁎
Wei Ding1, Jing Cui1, Yongteng Zhao, Benyong Han, Tao Li, Peng Zhao, Jun-Wei Xu, Xuya Yu
Faculty of Life Sciences and Technology, Kunming University of Science and Technology, Kunming, Yunnan, China

G R A P H I C A L A B S T R A C T

Stage I: Stage II:

mixotrophic photoautotrophic

Protein
Inoculating
Carbohydrate content

Autotrophic cells Glutamate

Adding
Biomass GABA

Induction culture by salt

FA
stress and high illumination

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

Keywords: The primary goal of this study was to assess the roles of chemical factors and bioprocess strategies on a mixo-
Haematococcus pluvialis trophic culture of the microalga Haematococcus pluvialis during γ-aminobutyric acid (GABA) production. A two-
γ-Aminobutyric acid stage strategy was used to increase the biomass and GABA accumulation of H. pluvialis. In stage I, mixotrophic
Two-step cultivation growth of H. pluvialis in the presence of fulvic acid (FA) produced a high biomass (1.84 g L−1) with a GABA
Fulvic acid
content of 25.45 mg g−1. Furthermore, a maximum GABA content of 38.57 mg g−1 was obtained when cells
Salt supplementation
were cultured with 0.4 g L−1 NaCl under photoautotrophic conditions in stage II, whereas the carbohydrate
content of cells sharply decreased from 26.68 to 18.22%. In addition, salt stress upregulated the expression of the
gad and cam genes. The results of this study demonstrate an efficient strategy to produce GABA from the mi-
croalga H. pluvialis.

1. Introduction inflammatory, antioxidant, antidiabetic, and nerve calming effects

γ-Aminobutyric acid (GABA) is a ubiquitous four carbon non-pro-
tein amino acid that has been observed in animals, bacteria, micro-
algae, and plants (Poojary et al., 2017; Brown, 1991). GABA is widely
used in the pharmaceutical and nutraceutical industries due to its many
biological and health-promoting functions, such as its diuretic, hypo-
lipidemic, neurotransmitter inhibition, anticarcinogenic, anti-
(Nikmaram et al., 2017; Dhakal et al., 2012).
GABA has been investigated in several organisms, including bac-
teria, algae, plants and animals (Michaeli and Fromm, 2015; Jantaro
and Kanwal, 2017). In plants, GABA biosynthesis occurs via the dec-
arboxylation of glutamate, which is catalysed by glutamate decarbox-
ylase (GAD) (Liao et al., 2017; Kim et al., 2016; Jantaro and Kanwal,
2017). GABA production was reported to be greatly enhanced

⁎
Corresponding author.
E-mail address: xuya_yu@163.com (X. Yu).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.biortech.2019.121334
Received 3 February 2019; Received in revised form 8 April 2019; Accepted 10 April 2019
Available online 11 April 2019
0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
W. Ding, et al.
accompanying the rapid increase in GAD levels under abiotic stresses,
such as mechanical injury, anoxia, heat or cold shock and salinity
(Kinnersley and Turano, 2000; Nicolas and Fromm, 2004). In previous
studies, GABA biosynthesis and metabolism were observed to be sti-
mulated by salt stress in Arabidopsis thaliana and tobacco (Renault et al.,
2010; Zhang et al., 2011). Nicolas et al. (2003) suggested that the
sudden increase in GABA accumulation after exposure to abiotic
stresses is an indication of its role in signalling in plants. Furthermore,
GABA is primarily synthesized through an irreversible reaction cata-
lysed by glutamate decarboxylase using glutamate as a substrate. Thus,
improving GAD activity can promote the accumulation of GABA (Shi
et al., 2016). In addition, a number of studies have shown that GAD
activity is stimulated by its binding to Ca2+/calmodulin (CaM) (Akçay
et al., 2012; Chevrot et al., 2006).
In contrast with higher plants that can accumulate GABA, micro-
algae feature numerous advantages, including rapid growth rate and
strong ability to fix and convert carbon dioxide (CO2) into biomass.
Furthermore, autotrophic microalgae can efficiently transform light
energy and inorganic nutrients into biomass that is rich in value-added
products, such as docosahexaenoic acid, pigments, proteins, amino
acids, and carbohydrates (Markou and Nerantzis, 2013). In particular,
certain cases are associated with microbial GABA allergy (Rashmi et al.,
2018). Therefore, GABA production from H. pluvialis will be safer and
more economic than that from microbes and plants. In addition, a
number of studies have demonstrated that salinity is a major factor
affecting the metabolism of microalgae, and salt has been widely used
to increase the production of value-added products in microalgae
(Markou and Nerantzis, 2013; Gao et al., 2015). Haematococcus pluvialis
is a unicellular green microalga that has been widely studied because of
its ability to accumulate a larger number of valuable products (Doria
et al., 2018; Ding et al., 2018a). Recent consumer necessity for food
products that are safe and “healthy” and exhibit added benefits (nu-
traceuticals/functional components) resulted in the exploration of
novel techniques to improve the content of bioactive compounds.
Hence, H. pluvialis can function as bio-based feedstock to help meet the
ever-increasing demands for natural products. However, the production
of GABA by microalgae has been rarely described (Filomena, 2013),
particularly for H. pluvialis.
The results of previous studies have suggested that the biosynthesis
of high value components (xanthophylls, astaxanthin and β-carotene) in
H. pluvialis is triggered when the cells are subjected to varieties of
stresses, such as nutrient starvation, high light intensity, and high
salinity (Zhao et al., 2019; Wu et al., 2013; Su et al., 2014). However,
the production of microalgal biomass is greatly inhibited under condi-
tions of environmental stress (Ding et al., 2018a; Shang et al., 2016).
Thus, a two-stage cultivation strategy has been developed to increase
biomass during the first the stage and accumulate metabolites during
the second stage (Wan et al., 2015). Previous studies have focused on
using an autotrophic-photoinduction two-stage cultivation strategy to
enhance biomass and generate astaxanthin (Shang et al., 2016). Goksan
et al. investigated the use of mixotrophic cultivation by the addition
sodium acetate (NaAc) to promote biomass generation and carotenoid
synthesis and observed an increase in biomass productivity and car-
otenoid concentration (Goksan et al., 2010). In addition, phyto-
hormones and growth regulators have been widely used to enhance
biomass (Zhao et al., 2019). Fulvic acid (FA) is a plant growth regulator
that enhances cell membrane permeability, photosynthesis, nutrient
assimilation, and respiration, as well as controls incretin levels and
increases the production of secondary metabolites (Çimri ̇ ṅ et al., 2010;
Che et al., 2016). Wang et al. (2018) observed that FA enhanced en-
hances growth and PUFA level of microalga, and Zhao et al. (2015)
reported that FA can improve biomass and astaxanthin accumulation in
H. pluvialis. However, no information has been reported regarding the
effect of NaAc combined with FA on H. pluvialis growth.
Although H. pluvialis is widely used in the production of astax-
anthin, its potential as feedstock to produce GABA has not been
2
Bioresource Technology 285 (2019) 121334
explored. In this study, a newfangled strategy to induce GABA bio-
synthesis was developed using a two-step cultivation and salt stress
process for GABA accumulation in H. pluvialis. The effect of NaAc
combined with FA and salt stress on the biomass and GABA accumu-
lation was investigated. Subsequently, the possible changes in the
physiology and biochemical characteristics were evaluated along with
the transcription of GABA biosynthetic genes of H. pluvialis. The results
of this study provide a foundation for the application of NaCl in GABA
production through a two-step H. pluvialis cultivation strategy.
Furthermore, this study provides valuable insights into the mechanism
of GABA biosynthesis in microalgae that may be useful for the devel-
opment of novel strategies for the hyperproduction of high value-added
secondary metabolites by microalgae.
2. Materials and methods
2.1. Microalga strain and cultivate condition
The H. pluvialis strain LUGU (18S GenBank: KM115647.1) was iso-
lated from water samples collected from Lake Lugu (27°42′00″N and
100°47′00″E) (Zhao et al., 2015). The strain was maintained and cul-
tured with a light intensity of 30 μmol m−2 s−1 in a bubbling column
photobioreactor (0.2 m diameter, 0.3 m height, for 3 L) containing 2 L
of BBM that was aerated with filtered air at a rate of 0.4 vvm. The
cultivation temperature was 25 ± 1 °C.
2.2. Two-step H. pluvialis culture conditions
For the first stage culture, the green vegetative H. pluvialis LUGU
strain was incubated at a density of ca. 2.5 × 105 cells mL−1 in a 1 L
Erlenmeyer flask containing 650 mL of fresh BBM with 2 g L−1 sodium
acetate (NaAc) and was aerated with filtered air at a rate of 0.4 vvm.
The cultivation temperature was 25 ± 1 °C, and the light intensity was
50 μmol m−2 s−1. In addition, 5 mg L−1 FA was added to enhance the
biomass yield, with each treatment conducted in three biological re-
plicates.
For the second-stage culture, algal cells from the stage I culture at 7
d were centrifuged at 3800×g for 5 min and washed with sterile water
to remove residual medium. The cells are resuspended in BBM and
exposed to a high illumination intensity of 150 μmol m−2 s−1 using a
white-light fluorescent lamp. The density of microalgae in the initial
culture was adjusted to 2.5 × 105 cells mL−1 and the temperature was
maintained at 28 ± 1 °C. To induce the production of GABA, the cells
were treated with different concentrations of NaCl (0, 0.2, 0.4 and
0.6 g L−1), where cells cultivated without NaCl in the medium were
used as a control. Each experiment was conducted in triplicate, with
samples withdrawn on a daily basis.
2.3. Measurements of biomass, glutamate, GABA and astaxanthin contents
To determine the biomass, 10 mL of algal culture was centrifuged
for 5 min at 3800×g. The pelleted cells were washed twice with sterile
water, after which the cells were collected and dried at −80 °C for 26 h
using a vacuum freeze dryer until a constant weight was obtained.
The glutamate and GABA contents of the cultures were determined
by high-performance liquid chromatography (HPLC) with a reverse-
phase C18 column (Waters, 25 cm × 4.6 mm) and a photodiode array
detector (Waters 996, USA) (Syu et al., 2008). For each sample, 100 mg
of dry algal cells was ground thoroughly with quartz sand. The powder
was then extracted with a 4% acetic acid aqueous solution and cen-
trifuged for 15 min at 6037×g. Next, 4 mL of ethyl alcohol was added
and the sample was centrifuged for 20 min at 16 770×g to remove
macromolecular biomolecules. The purified supernatant was thor-
oughly dried in a vacuum drying oven at 45 °C to volatilize the ethanol
and acetic acid. The leftover material was dissolved with 0.5 mL of
sterile water and centrifuged for 10 min at 2683×g.
W. Ding, et al. Bioresource Technology 285 (2019) 121334

The supernatant liquid was filtered using a 0.45 μm membrane

filter, and 100 μL of the filtered liquid was assayed by HPLC. The
standard GABA solution, glutamate (Sigma) and the samples were
analysed by pre-column derivatization of phenylthiocarbamyl-GABA
(PTC-GABA) from phenyl-isothiocyanate (PITC). Mobile phase A
(0.5 mL triethylamine, 0.7 mL acetic acid, 8.205 g sodium acetate, and
5.0 mL acetonitrile, with the final volume brought up to 1000 mL with
water); and mobile phase B (acetonitrile-water (3: 2) were used for
isocratic elution at a flow rate of 0.6 mL min−1 for the entire run.
Twenty microliters of each sample were injected and detected at
254 nm, with the column temperature set at 27 °C.
Weighed dried cell samples were used for astaxanthin analysis using
a method described earlier (Ding et al., 2018a).

2.4. Quantification of the chlorophyll, carbohydrate and protein contents of

H. pluvialis
To evaluate cellular physiology of H. pluvialis, i.e., the chlorophyll,
carbohydrates, and proteins contents, the cells from 5 mL of algae
culture from the second stage were collected by centrifugation. The
chlorophyll content was calculated from the absorbance of the extract
at 645 and 663 nm, as described by Wellburn (1994). The total protein
content in the microalga was evaluated according to Bradford, using
bovine serum albumin as standard (Berges et al., 1993). Lyophilized
algal powders were used to analyse the total carbohydrate content (Ma
et al., 2016).
2.5. RNA isolation, cDNA synthesis and gene expression analysis
Total RNA was isolated from H. pluvialis as previously described
(Ding et al., 2018b), and cDNA was synthesized using an RT-PCR kit
(TaKaRa; Shanghai, China) according to the manufacturer’s instruc-
tions. The mRNA levels of the gad, cam and 18s genes were analysed
with an ABI 7500 Real-Time PCR System with SYBR Green Master Mix
(TaKaRa; Shanghai, China), using the primers listed in the
Supplementary data. The expression levels were normalized against the
H. pluvialis 18S rRNA gene (an internal control), which was expressed at
a constant level under all experimental conditions. The relative gene
expression was determined according to the 2−ΔΔCT method (Livak and
Schmittgen, 2001).
2.6. Statistical analyses
Each experiment was performed at least three times. All data were
statistically analysed using Student’s t-test. The results were considered
significant at p < 0.05 using a two-tailed analysis.
3. Results
3.1. Effect of FA on biomass in the stage I culture
To enhance the biomass of H. pluvialis, exogenous FA was added
during the stage I culture. As shown in Fig. 1, the biomass of H. pluvialis
obtained in the FA treatment groups was higher than that obtained
from the control, with the FA-supplemented groups exhibiting a high
growth rate. The highest biomass reached 1.84 g L−1 in the 5 mg L−1
FA treatment group, which was 17.19% higher than that of the control
(1.57 g L−1). After 9 days of cultivation, the biomass obtained in the 3
and 7 mg L−1 FA groups was 1.72 and 1.64 g L−1, respectively
(Supplementary data). These results suggest that the addition of FA
addition in combination with NaAC is an effective strategy for to in-
crease the production of H. pluvialis biomass.
Fig. 1. Effect of fulvic acid (FA) treatments on the biomass of H. pluvialis during
mixotrophic cultivation. Vertical bars represent the means ± SD (n = 3). *
indicates statistical significance at p < 0.05, ** indicates statistical significance
at p < 0.01 compared with the control.
3.2. Effect of salt stress on the biomass, GABA and astaxanthin content of
H. pluvialis during stage II
After stage I growth, the algal cells were cultured under stage II
conditions to promote GABA production. In this stage, different salt
concentrations were used to induce the accumulation of GABA. Fig. 2A
shows the H. pluvialis growth curves cultured in the medium supple-
mented with NaCl at concentrations of 0 (control), 0.2, 0.4 and
0.6 g L−1 for 4 days under high light intensity conditions. Different
growth profiles were observed for the various treatments for stage II.
After three days of cultivation, the biomass in the 0.2 and 0.6 g L−1
NaCl groups reached approximately 0.73 and 0.69 g L−1, respectively.
The biomass of H. pluvialis exposed to 0.4 g L−1 NaCl (0.76 g L−1) was
the highest compared with other groups, although it did not sig-
nificantly differ from the control (0.74 g L−1).
The GABA content in the microalgal cells increased significantly in
response to the NaCl addition (Fig. 2C), gradually increasing during the
0.4 g L−1 NaCl treatment and reaching the highest content
(38.57 mg g−1) after 3 d, representing a nearly 1.25-fold increase over
the untreated control (30.95 mg g−1) after 2 d. In the 0.6 g L−1 NaCl
group, the GABA content showed a rapid increase from 25.45 to
33.61 mg g−1 after a day of cultivation and then showed a downward
trend. In the 0.2 g L−1 NaCl group, the GABA content slightly increased,
peaking at 32.04 mg g−1 after 2 days of cultivation.
Remarkable differences in astaxanthin content between non-treated
and NaCl-treated groups were observed under high light and salt stress
conditions (Fig. 2D). The astaxanthin content of cells treated with 0.2,
0.4, and 0.6 g L−1 NaCl was significantly higher than that in the control
group and increased by 42.74%, 21.08%, and 21.37% on the fourth
induction day, respectively. The maximal astaxanthin content
(5.01 mg g−1) occurred in the 0.2 g L−1 NaCl treatment. Thus, although
NaCl caused no positive effect on biomass, its addition improved the
accumulation of GABA and astaxanthin.
3.3. Changes in the glutamate content of H. pluvialis during GABA
accumulation
As a precursor of GABA biosynthesis, glutamate is transformed to
GABA by GAD. As shown in Fig. 2B, the content of glutamate was ex-
hibited a concomitant decrease as the GABA level increased (Fig. 2C).
The glutamate level decreased sharply in H. pluvialis at 3 d in the
0.4 g L−1 NaCl treatment group, representing an approximately 45.53%
reduction relative to the content observed after 2 d, before increasing
after 4 d. The glutamate level in the 0.6 g L−1 NaCl group decreased

3
W. Ding, et al.
Fig. 2. (A) Effect of NaCl on the biomass of H. pluvialis during induction. (B) Effect of NaCl on the glutamate content of H. pluvialis during induction. (C) Effect of NaCl
on the GABA content of H. pluvialis during induction. (D) Effect of NaCl on the astaxanthin content of H. pluvialis during induction. Vertical bars represent the
means ± SD (n = 3). * indicates statistical significance at p < 0.05, ** indicates statistical significance at p < 0.01 compared with the control.
gradually over the course of the culture period. Moreover, the gluta-
mate content exhibited almost the same decreasing trend in both the
control and 0.4 g L−1 NaCl groups. Taken together, with the accumu-
lation of GABA, the glutamate content is bound to decrease.
3.4. Physiological response of H. pluvialis to salt supplementation
Chlorophyll content in the NaCl-treated and control groups ex-
hibited similar trends after being cultured in the presence of NaCl
(Fig. 3A). The chlorophyll content increased in response to salt sup-
plementation. Maximum chlorophyll contents of 16.05, 18.00, and
21.49 mg g−1 were observed in cells treated with 0.2, 0.4, and 0.6 g L−1
NaCl, respectively. After peaking, the chlorophyll concentration in the
NaCl-treated groups gradually decreased. Concurrently, the chlorophyll
concentration in the control group continually decreased.
The content of carbohydrate was also determined in H. pluvialis
during NaCl-treatment. The results presented in Fig. 3B show that the
carbohydrate level rapidly increased from 16.15 to 33.26% for the first
2 days of growth in the presence of 0.2 g L−1 NaCl. The highest cellular
carbohydrate content was observed in the control, reaching to 30.86%
after 3 d. In contrast, in the 0.4 and 0.6 g L−1 NaCl groups, the highest
carbohydrate content observed was 26.68 and 25.83%, respectively.
Afterwards, the carbohydrate content exhibited a continual decrease.
Remarkably, the carbohydrate content in the 0.4 g L−1 NaCl group re-
mained at a low level compared with the other groups before the end of
the three-day culture period.
The synthesis of protein in H. pluvialis cells increased from the stage
I culture to the NaCl treatment in stage II and in the control group
(Fig. 3C). Under the 0.4 and 0.6 g L−1 NaCl treatments, the protein
content was enhanced to 30.05 and 29.30% on day 1. Moreover, the
protein content increased to 27.65 and 29.05% in the 0.6 g L−1 NaCl
and control samples, respectively. Similar to the chlorophyll and car-
bohydrate contents, after reaching the maximum levels the protein
4
Bioresource Technology 285 (2019) 121334
contents began to gradually decrease in both the NaCl-treated and
control groups.
3.5. GABA biosynthesis-related gene expression associated with NaCl stress
in H. pluvialis
To determine whether the transcription of genes related to GABA
synthesis was induced by salt stress under high light conditions, the
expression patterns of two primary GABA biosynthesis genes (cam and
gad) were determined by qRT-PCR. As shown in Fig. 4, cam and gad
expression increased gradually in the 0.4 g L−1 NaCl group, increasing
significantly after 2 and 3 d by 5.55- and 4.88-fold compared to the
control, respectively. The expression of cam and gad in the 0.4 g L−1
NaCl group showed a slight upregulation, with maximum increases of
2.57- and 2.98-fold higher than that of the control, respectively. In
addition, the peak expression of cam and gad in the 0.6 g L−1 NaCl
treatment group was 3.37-, and 2.59-fold higher, respectively, than
those of the control group during the cultivation period. These results
agree with the increased GABA content observed during the culture
process (Fig. 2C).
4. Discussion
Microalgae have gained a great deal of interest because they are a
natural source of biofuels and biopharmaceuticals. There are many
strategies to promote microalgae biomass and metabolite production
based on controlling culture conditions, one of which is the addition of
phytohormone to enhance microalgal secondary metabolite production
by regulating biochemical pathways (Ding et al., 2018b). In this study,
the ability of FA to increase the biomass of H. pluvialis was tested under
mixotrophic conditions. The results showed a biomass concentration in
the 5 mg L−1 FA-treated group, that is, an increase of 17.19% compared
with that of the control culture (Fig. 1), similar to the results of several
W. Ding, et al. Bioresource Technology 285 (2019) 121334

Fig. 3. Effect of NaCl on biochemical composition of H. pluvialis. (A) Chlorophyll content, (B) carbohydrate content, (C) protein content. Vertical bars represent the
means ± SD (n = 3). * indicates statistical significance at p < 0.05, ** indicates statistical significance at p < 0.01 compared with the control.

studies, which showed that FA can enhance the biomass of microalgae,
such as Monoraphidium and H. pluvialis (Che et al., 2016; Zhao et al.,
2015). FA is a growth regulator that stimulates an increase in photo-
synthesis and carbon and oxygen metabolism. In addition, FA plays an
important role in plant signal transduction (Che et al., 2017), which
may be explained by a variety of regulatory functions similar to hor-
mones. However, microalgal metabolism at the biochemical and mo-
lecular levels after FA treatment still requires further research.
Salt has been extensively used to induce the biosynthesis of a
number of high value metabolites in microalgae (Jiang and Chen, 1999;
Gao et al., 2015). Thus, during stage II growth, H. pluvialis cells were
treated with salt to improve GABA accumulation. The biomass of the
cells grown in the presence of 0.4 g L−1 NaCl was not significantly
different to that of the control, but the salt treatment did improve the
accumulation of GABA (Fig. 2A and C). A similar phenomenon was
observed in previous study, where an increase in concentration and
synthesis of GABA was promoted with the addition of ethephon in
Chlorella vulgaris, whereas the biomass remained largely unchanged
(Kim et al., 2016). Kempa et al. (2008) also observed that the GABA
content of Arabidopsis thaliana increased after NaCl treatment.
Moreover, the GABA production observed with addition of NaCl in
H. pluvialis cultured under high light intensity was much higher than
that observed in other studies of GABA biosynthesis. For instance,
Grewal et al. investigated GABA biosynthesis from Lactobacillus brevis
by solid-state fermentation using toxic deoiled cottonseed cake as
substrate, and the highest GABA content of 19.7 mg g−1 cottonseed
cake was obtained on the 6th day of culturing (Grewal and Khare,
2017). Additionally, the GABA content obtained under anoxic condi-
tions in Tea (Camellia sinensis L.) was observed to gradually increase,
peaking at 0.73 mg g−1 fresh weight (Liao et al., 2017). However, due
to differences in species and cultivation conditions, the dependence of
regulators on the species and cultivation modes makes our results

Fig. 4. Relative gene transcription levels involved in GABA biosynthetic pathways of H. pluvialis under salt stress and photoinduction condition. (gad: glutamate
decarboxylase; cam: calmodulin).

5
W. Ding, et al.
incomparable with those of other studies, and further experimental
verification is required.
In this study, the astaxanthin content has also been examined.
Fig. 2D shows the remarkable enhancement of astaxanthin accumula-
tions when cells were cultured with NaCl supplementation under pho-
toautotrophic conditions in Stage II. The exploitation of cultivated H.
pluvialis along with the accumulation of astaxanthin and GABA will
strongly enhance a bio-based economy.
In plants, GABA is considered a temporary nitrogen storage source
as it is produced from glutamate under stressful conditions. Therefore,
the experimental groups should implement the BBM medium exogenous
addition of NaNO3 (BBM + N) and BBM medium deficiency of NaNO3
(BBM-N) (Supplementary data). Unlike the nitrogen-deficient treat-
ment, despite the remarkably increased GABA content in H. pluvialis
due additional nitrogen source, the astaxanthin content in nearly all the
experimental groups, except in nitrogen deficiency (ND) group, showed
no significant increase. However, in this study, a maximum GABA
content of 38.57 mg g−1, which was higher than that of the optimal
NaNO3 treatment group, was obtained when cells were cultured with
0.4 g L−1 NaCl, and the astaxanthin content also significantly increased.
These results showed that abiotic stress is a more effective strategy than
nitrogen-rich culture in promoting the accumulation of GABA by H.
pluvialis. This result is similar to that of previous studies, that is, the
accumulation of GABA can be enhanced by different environmental
stresses (Akçay et al., 2012; Bouché et al., 2003).
Simultaneously, the present work observed that salt supplementa-
tion resulted in a rapid modification of the chlorophyll, carbohydrate
and protein levels of H. pluvialis. After treating the microalgae with
NaCl, the levels of chlorophyll were significantly altered, indicating an
increase in microalgal metabolism. H. pluvialis primarily relies on
photosynthesis to fix carbon and for the transfer of carbon-containing
components into various metabolic routes for the synthesis of major
macromolecules. With the addition of NaCl, the microalgal carbohy-
drate levels increased rapidly in the early stage of cultivation and then
began to decrease until it was lower than that of the control group
(Fig. 4B). Carbohydrate stored in cells are essential for carbon skeletons
and as an energy supply. GABA metabolism plays an important role in
nitrogen and carbon metabolism. The sharp increase in the content of
GABA as a result of NaCl supplementation is in agreement with the
putative role of GABA in responding to changes in the environment. The
consumption of carbohydrates may be explored by following the ac-
cumulation of GABA, and enhanced carbohydrate degradation provides
carbohydrate skeletons and energy to adapt to changes in culture
conditions. Kempa et al. (2008) observed that in Arabidopsis thaliana
under salt stress conditions, a gradual reduction in the starch level was
accompanied by an enhancement in GABA synthesis.
As a structural material and a bioactive macromolecule in cells,
proteins are very sensitive to changes in environmental conditions
(Araújo et al., 2011; Brikis et al., 2018). A significant increase in protein
levels was observed upon subjecting H. pluvialis cells to salinity con-
dition after 1 d, after which the protein level in the salt-treated cells
decreased and was lower than that of the control cells (Fig. 3C). This
result is consistent with the findings of another study where it the level
of protein was shown to rapidly increase in Monoraphidium cells ex-
posed to high illumination conditions for several days and then gra-
dually decreased (Li et al., 2017). Thus, the levels of GABA exhibited a
coordinated increase that may be the result of enhanced protein cata-
bolism and/or re-allocation of nitrogen caused by stressful conditions.
A similar response to changing levels of amino acids was observed in
Populus euphratica, suggesting this response is similar to that observed
for high salinity (Brosché et al., 2005).
Salinity has a strong effect on gene expression (Kilian et al., 2007).
Salt stress regulates the expression of several genes encoding enzymes
involved in GABA metabolism. The protein encoded by the gad gene
catalyses the transformation of glutamate to GABA, a crucial and rate-
limiting step in GABA synthesis. After NaCl treatment, the transcription
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Bioresource Technology 285 (2019) 121334
of gad was gradually upregulated, promoting the catalysis of glutamate
into GABA. Liao et al. (2017) observed a similar result where gad up-
regulation was observed to improve the catalysis of the decarboxylation
of glutamate to GABA in tea (Camellia sinensis l.) under anoxic condi-
tions. Glutamate that is the key precursor in the GAD pathway and
plays a central role in carbon and nitrogen metabolism, specifically in
ammonia and nitrate/nitrite assimilation in green algae (Jantaro and
Kanwal, 2017). On day 3, with the upregulation of gad transcription,
the glutamate levels exhibited a sharp decrease, whereas the levels of
GABA levels to greatly increase (Fig. 2B and C). GAD proteins generally
contain a C-terminal region known as the Ca2+/calmodulin (CaM)
binding domain, which autoinhibits the activity of this enzyme. How-
ever, the autoinhibition is relieved by the binding of Ca2+/CaM to this
domain (Takayama et al., 2017). In this study, the transcription of cam
was upregulated after the NaCl treatment, which is consistent with the
activation of GAD during cultivation and indicating that the tran-
scription of cam may regulate the activity of GAD, affecting GABA ac-
cumulation in H. pluvialis. Takayama et al. showed that Ca2+/CaM
confers GAD activation (Takayama et al., 2017), and Bouché et al.
(2003) proposed that CaM may also participate indirectly in managing
reactive oxygen species levels through CaM-regulated GABA synthesis
and the GABA shunt metabolic pathway. In addition, Chauhan et al.
(2017) reported that Ca2+/CaM is important in signalling, regulating
the gene expression of plants in stress signalling.
5. Conclusions
H. pluvialis was used for production of GABA via a two-step culti-
vation process. An FA concentration of 5 mg L−1 was optimal for mix-
otrophic cultivation of H. pluvialis with 2 g L−1 NaAc as the organic
carbon source, resulting in an increased biomass that was 17.19%
higher than that of the control. A novel two-stage strategy of mixo-
trophic cultivation with NaAc and FA and autotrophic cultivation with
NaCl significantly enhanced the GABA content from 25.45 to
38.57 mg g−1. Thus, the two-stage strategy appears to be an appro-
priate approach for improving GABA accumulation by H. pluvialis
during mixotrophic cultivation.
Acknowledgments
This work was funded by the National Natural Science Foundation
of China (21766012).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.biortech.2019.121334.
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