Mal J Nutr 29(3): 453-466, 2023

The effect of photoperiodism on nutritional potency of

Euglena sp. Indonesian strains
Khusnul Qonita Maghfiroh1, Tia Erfianti1, Istini NurAfifah1, Ria Amelia1, Dedy
Kurnianto2, Brilian Ryan Sadewo3, Revata Maggandari4, Bambang Retno Aji1,
Arief Budiman3 & Eko Agus Suyono1*

1
Faculty of Biology, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia;
2
Research Centre for Food Technology and Processing, National Research and
Innovation Agency, Yogyakarta 55861, Indonesia; 3Department of Chemical
Engineering, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia; 4Master in
System Engineering, Faculty of Engineering, Universitas Gadjah Mada, Yogyakarta
55281, Indonesia

ABSTRACT

Introduction: Biomass microalgae functional materials, such as drugs and food

supplements, have recently received much attention. Euglena sp. is a particularly
appealing microalgae because of its simplicity of culture and capacity to produce a
wide range of bioactive compounds. Moreover, it is one of the few microorganisms
that produces carbohydrate, lipid, protein, ß-1,3-glucans, antioxidants, phytotoxins,
wax esters, and polyunsaturated fatty acids that can be used to make nutraceuticals,
pharmaceuticals, and cosmeceuticals. However, the potential utilisation of Euglena
sp. for production of food supplements has been exploited only on a limited basis.
Methods: This study was modified by adding protocatechuic acid and photoperiodism
for 12:12; 14:10; 16:8; and full dark to affect the metabolite content of Euglena
sp. Results: Results showed that the photoperiod had significant effect on lipid,
chlorophyll-a, and carotenoid levels in the control treatment, with the highest levels
as follows: 0.52±0.03 g/L, 1.20±0.01 x10-2 g/L, 0.30±0.02 x10-2 g/L; while the others
were not significantly affected by the treatment, with the highest protein content at
full dark 3.10±0.2 x10-2 g/L; chlorophyll-b at photoperiod 14:10 0.70±0.03 x10-2 g/L;
paramylon at photoperiod 12:12 1.90±0.02 x10-1 g/L. The highest carbohydrates
were found in control, with a level of 1.20±0.02 g/L. Conclusion: Photoperiodism
is recommended to enhance productivity of protein, paramylon, and chlorophyll-b,
while full light is recommended to enhance carbohydrate, lipid, chlorophyll-a, and
carotenoid production in Euglena sp. to improve the quality of food nutrition.

Keywords: Euglena sp., photoperiod, protocatechuic acid

INTRODUCTION friendly and renewable. In addition,

recent advances in biological engineering
In recent years, it has been known that
and multi-omics have revealed numerous
scientists are interested in developing the
potentials for microalgae bioproducts in
use of microalgae biomass as a source
the nutrition, media, pharmaceutical,
of component for producing bioactive
and commodity industries (Bajhaiya,
metabolites that are environmentally

__________________________
*Corresponding author: Dr Eko Agus Suyono, M.App.Sc
Faculty of Biology, Gadjah Mada University, Yogyakarta, Indonesia
Tel: (0274) 580839 E-mail: eko_suyono@ugm.ac.id
doi: https://doi.org/10.31246/mjn-2023-0004
454 Khusnul QM, Tia E, Istini N et al.

Moreira & Pittman, 2017; Stengel & Changes in photoperiod can affect
Connan, 2015). Euglena (protist) is the production of total pigment, fatty
a flagellated microfactory organism acid, and protein content in Chlorella
living in autotrophic, heterotrophic, or vulgaris, lipid formation and growth
mixotrophic environments. It can grow in Porphyridium cruentum (Khoyi et
heterotrophically with various carbon al., 2009; Oh et al., 2009), as well
sources and pH levels in wastewater as nutrient utilisation and biomass
(Mahapatra, Chanakya & Ramachandra, in Chaetoceros muelleri production
2013). Euglena sp. is not like bacteria or (Minggat, Roseli & Tanaka, 2020). The
protozoa, so it does not have a dangerous duration of photoperiod significantly
or pathogenic risk to other living things, impacts diatom cell development (dark:
especially humans, and their numbers light cycle) (Palanisamy et al., 2022).
can be increased easily. Light intensity greater than 150 E m-2s-
Euglena is high in carbohydrate-active 1
is thought to inhibit cell development;
enzymes, with an exceptional ability to it may oversaturate cell growth under
synthesise complex carbohydrates for more extended irradiation, as indicated
a unicellular organism. Furthermore, by the difference between the dark:light
Euglena cells contain many of nutrients cycles of 8:16 and 16:8 (Li, Talmy &
like fatty acids, such as eicosapentaenoic Campbell, 2017). Therefore, to confirm
acid, docosahexaenoic acid, and the potential of Euglena sp. as a source
vitamins, implying the genus is a of various bioactive for human nutrition,
valuable and potential resource for food various cultivation techniques can be
supplement. Euglena sp. also contains carried out, one of which is by using
many nutrients, such as carbohydrates, different photoperiods.
lipids, proteins (primary metabolites),
ß-1, 3-glucans (paramylon), carotenoids, MATERIALS AND METHODS
tocopherol, essential amino acids,
Isolation & cultivation
minerals, phycobiliproteins (PBPs),
Water samples for microalgae isolation
phytohormones, phytosterols, phenolic
were collected from the Dieng Plateau,
compounds, and mycosporine-like amino
Wonosobo, Middle Java, Indonesia
acids (secondary metabolites), that have
and isolated using capillarity pipette
been shown to possess nutritional,
methods in aseptic laminary air flow.
antioxidant, neuroprotective, anti-
Cramer–Myers (CM) medium was
inflammatory, antimicrobial, anti-
used as a growth medium, with the
angiogenic, and anti-cancer properties
composition (g.L-1): 1 gram NH4SO4, 1
(Haque et al., 2014; Rico et al., 2017;
gram KH2PO4, 0.2 gram MgSO4.7H2O,
Singh et al., 2017; Nakazawa, 2017).
0.02 gram CaCl2.2H2O, 100 µL trace
Therefore, the use of Euglena sp. as a
metal mix, 100µL Na2MoO4.2H2O
healthy supplementary food is promising.
solution, 20 µL vitamin B1 solution, 25
In the cultivation of Euglena sp.,
µL vitamin B12 solution, with a pH of
dark:light cycle influences its spectral
5.5 and an additional carbon source of
composition, oscillation pattern, and
0.8 g protocatechuic acid (PCA). The CM
photoperiod, all of which contribute
medium was previously sterilised using
significantly to the microalgae
an autoclave at 121°C for 20 minutes.
metabolic process. Optimal irradiance,
After making the media, the starter
oscillation pattern, and dark length
was left for two weeks to equalise the
all influence phytoplankton metabolic
age of Euglena sp. The starter was then
activity (Oostlander et al., 2020).
transferred to a 50 mL culture bottle and
 The effect of photoperiodism on Euglena metabolite 455

the volume of CM was 450 mL; thus, the was calculated using the following
total in one culture bottle was 500 mL. formulas (equations 3 and 4), where X
is cell density, X0 is initial cell density,
Identification and screening strain Xmax is maximum cell density, and
Samples from various strains that were max is maximum specific growth rate
successfully isolated were observed (Phukoetphim et al., 2017).
under the Olympus CX22LED1000x
magnification light microscope, with
added immersion oil and connected with
dx
dt = µmax ( 1 – μx ) x (3)
max

Optilab Advance Observer (MTN034).

Strain screening was done by cultivating
isolates that have successfully grown
without contamination, namely seven
x = ( 1 – [(xxoxo.exp(µ t)
max

) (1 – exp(µ max t) )]
) (4)
max
strains out of a total of 35 strains.
The seven lines were IDN 23, IDN 29, The parameters in the Gompertz model
IDN Mix, IDN 33 A Aerobic, IDN 33 A were maximum cell production (rm) and
Anaerobic, IDN 33 B Aerobic, and IDN lag time (tL). The model was determined
33 B Anaerobic. Then, their growth rates using the following formulas (equations
were compared, and the highest growth 5 and 6), where SSR is the sum square
rate was selected as the best candidate residual and SST is the sum square total
line. Microalgal growth was measured (Phukoetphim et al., 2017).
every day. In addition, the concentration rm.exp(1)
of microalgae cells was determined x = Xo + [Xmax.exp [– exp (( xmax ) (tl – t) + 1] (5)
by measuring the culture’s optical
density (OD) with the Thermo Scientific SSR
Evolution 201 UV-Vis spectrophotometer R 2 – (1 – ) (6)
SST
at an absorbance of 680 nm in three
replicates. Calculation of cell-specific Determination of cell growth
growth rate was done using the following Cell growth in different photoperiods
formulas (1,2): was compared by counting cells every
24 hours using a light microscope and
Ln (Nt – N0) Haemocytometer Neubauer 1 mm. After
µ= (1)
t1 – t0 shaking the sample to homogenise it, 100
µL was pipetted into a 2 mL microtube
Ln2 using a micropipette. The model was
Dt= (2)
24xμ then transferred to a haemacytometer
and the cells were counted using a
µ=Specific growth rate; Dt=generation light microscope linked to a computer
time (hours); Nt=cell population on running optilab software. The number
the t-day exponential phase (cells of cells in each of the four corners was
mL-1); N0= cell population on day 0 of calculated and the total number of cells
the exponential phase (cells mL-1); t1-t0 was computed.
= time interval in the exponential phase
(days). Biomass calculation of Euglena sp.
Biomass production was calculated
Growth kinetics modelling every three days using the dry weight
The Logistic and Gompertz models were of cells. Two mL of sample culture was
used to model the growth kinetics of transferred into a 2 mL microtube. For
Euglena sp. First, the Logistics model
456 Khusnul QM, Tia E, Istini N et al.

10 minutes, the sample was centrifuged standard Bovine Serum Albumin (BSA)
at 4000 rpm. The supernatant was protein solutions from Abbkine kit at 20,
removed and washed with distilled 50, 75, and 100 µg/ml concentrations.
water. Cell suspensions at the bottom of
microtubes were dried in an incubator Percentage and productivity
oven at 37°C until they had a constant determination of Euglena sp.
weight. The final biomass was calculated primary metabolites
by subtracting the sample’s final weight Productivity measurements of primary
from its initial weight and then dividing and secondary metabolites were
it by its initial volume. calculated based on the following
equations (8,9) (Chen et al., 2020).
Carbohydrate estimation of
Total cell compound
Euglena sp. % cell compound =
Biomass
x 100% (8)
Dubois’s method quantified the
Productivity = Biomass productivity x % cell compound (9)
carbohydrate content in microalgae
biomass using phenol-sulfuric acid.
After creating a standard curve of Pigmentation analysis
carbohydrate concentrations, the A spectrophotometric method was used
sample’s absorbance was measured to determine pigment content. Two ml
using a spectrophotometer at 490 nm to culture was centrifuged at 4000 rpm for
determine carbohydrate concentration. 5 minutes. Then, the supernatant was
removed and the pellet was extracted
Lipid estimation of Euglena sp. overnight in the dark at 4oC with 1.5
The Bligh and Dyer method was used mL methanol (99.9%). The use of waves
to quantify the lipid content of Euglena on the spectrophotometer ranged from
sp. Extraction method that included a 400-750 nm. The concentrations of
1:2 ratio of chloroform and methanol, chlorophyll-a (Chl-a), chlorophyll-b
followed by a 1:1 ratio of chloroform (Chl-b), and photoprotective carotenoid
and aquades was used. The solution (PPC) were calculated using the following
was then centrifuged until three layers equations:
were formed, and the bottom layer was
removed and incubated in an oven at Chl a (µg/mL) = -8.0962 x λ652 + 16.5169
30°C for 24 hours. λ665;

Protein estimation of Euglena sp. Chl b (µg/mL) = 27.4405 x λ652 – 12.1688

The Bradford method was used to x λ665;
determine the protein content of
Euglena sp. This method was carried Total Carotenoids (µg/mL) = 4 x λ480
out by centrifuging the supernatant
from the separation process and adding Paramylon extraction and analysis
in a SDS solution. Following a 5-minute A culture of 10 mL Euglena sp. was
incubation at 95°C, a 5-minute taken and centrifuged at 4000 rpm
incubation at 4°C was performed. for 10 minutes. The supernatant was
Bradford’s solution was then added to discarded to obtain pellets. The pellets
the incubation samples. Absorbance were dissolved in 1% (w/v) SDS and
was then measured at 595 nm using 5% (w/v) Na2EDTA, then incubated in
an eLISA Reader Biotech. Protein a water bath at 37oC for 30 minutes.
content was calculated using standard The treatment was repeated without
linear curve regression equations from incubation in SDS-Na2EDTA solution
 The effect of photoperiodism on Euglena metabolite 457

and then the paramylon was washed called euglenoid movement, as well as
twice with distilled water. Paramylon having eye spots (stigmas) containing
pellets were dissolved in 2 mL NaOH. carotenoids that control the intensity of
The phenol sulfuric acid method light (Erfianti et al., 2023). Euglena also
was used to determine paramylon has a cell size ranging from 31-68 μm,
concentration. The extraction solution flagella for swimming, and a reservoir
was mixed in a test tube with 5% phenol (Al-Ashra, Abiad & Allahem, 2014).
and H2SO4 (sulfuric acid). The test tube Therefore, Euglena sp. was isolated
was allowed to stand for 10 minutes in and identified using a light microscope
a standing position. Next, the solution from all strains (Figure 1). The solitary
was vortexed for 30 seconds before being Euglena sp. measured ±50 µm in length.
allowed to stand at room temperature Microscopical analysis revealed that
for another 20 minutes. Then, a 490 Euglena cells were solitary and free to
nm spectrophotometer solution was swim. They typically lacked a cell wall
used. The paramylon standard was used and were elongated and spindle-shaped
to create standard curves. Paramylon with tapering ends. Seven different
productivity (g L-1 day-1) = last exponential strains (IDN 23, IDN 29, IDN Mix, IDN
phase-first exponential phase (Zhu & 33 A Aerob, IDN 33 A Anaerob, IDN 33
Wakisaka, 2018). B Aerob, and IDN 33 B Anaerob) were
obtained based on the isolation process,
Statistical analysis which were then screened by growing
All experiments were done in triplicates the isolation results in laboratory-scale
and data were shown as mean values CM medium to get the strain type with
of the three replicates. The various the best growth rate.
experiments’ mean values and standard
deviations were evaluated using
Microsoft Excel 2007. Using IBM SPSS
Statistics for Windows version 26.0 (IBM
Corp., New York, United States), the
results of each analysis were analysed
using one-way analysis of variance
(ANOVA) by rank at a 95% confidence
level.

RESULTS
There were many species of Euglena in
the Dieng plateau. Erfianti et al. (2023)
stated that Euglena sp. is a member of the
Euglena genus that has been successfully
isolated under extreme conditions.
The pH level of Dieng Peatland is 2.0-
3.5, suitable for the growth of Euglena. Figure 1. Cell of Euglena sp isolated
The special characteristics of Euglena from Dieng Plateau. Magnification 100 x
are elongated oblong or spherical
shaped cells, green in colour because Table 1 and Figure 2 showed that
they contain the pigment chlorophyll-a the highest biomass productivity was
and -b, and has a pellicle structure found in strain IDN 33 A Aerobic, with a
that allows its cells to make changes value of 0.76 g/L/day. The same results
458 Khusnul QM, Tia E, Istini N et al.

Table 1. Screening productivity of biomass Euglena sp.

Strain SGR (µ) DT (day-1) Productivity biomass (g/L/day)
IDN 23 0.149 0.193 0.614
IDN 29 0.153 0.189 0.668
IDN MIX 0.125 0.230 0.459
IDN 33 A Aerobic 0.168 0.172 0.764
IDN 33 A Anaerobic 0.122 0.238 0.450
IDN 33 B Aerobic 0.158 0.183 0.636
IDN 33 B Anaerobic 0.155 0.187 0.591

were obtained in measuring cell density day 13, while the control group started
using the spectrophotometric method. on day 15.
The results showed that strain IDN 33 Based on the one-way ANOVA
A Aerobic had a growth phase close to test, the photoperiod treatment of the
the previously optimised IDN 29 strain. control Euglena sp. had no statistically
It is important to note that variation in significant effect on the growth rate
microalgae can be seen between distinct of Euglena sp. (p=0.440). In this
genera, various species, and even strains experiment, higher carbohydrate
of the same genus (Taleb et al., 2016). content was obtained using a medium
From the two measurement processes, with dark condition and control
IDN 33 A Aerobic was then declared to treatment on day 9 at 1.10±0.02 g/L
be the selected strain that was to be and 1.20±0.02 g/L, respectively (Figure
used for the cultivation stage, with the 3a), with carbohydrate productivity of
highest specific growth rate value of 0.64x10-1 g/L/day and 1.11x10-1 g/L/
0.168 µ and the lowest doubling time of day, respectively. Moving to the following
0.172 day-1. graph (Figure 3b), higher lipid content
The optical density of Euglena sp. was obtained using a medium with
cultivated in CM medium and the dark condition on day 12 and control
addition of PCA combined with specific treatment on day 15 at 0.49±0.05 g/L
photoperiod modes is presented in and 0.52±0.03 g/L, respectively. Total
Figure 2. Optical density (OD) formed lipid content of Euglena sp. in full dark
a different pattern between the control treatment increased from 14.47% to
group without adding PCA and using 62.98%, while lipid content of Euglena
the full light treatment with the addition sp. control increased from 10.70% to
of PCA and photoperiod. On day 2, the 43.94%, with lipid productivity of 0.32
treatment group experienced a relatively x10-1 g/L/day and 0.26x10-1 g/L/day,
rapid increase in OD compared to the respectively. A higher protein content
control group. The treatment group was obtained using a medium with dark
entered the log phase faster than the condition on day 15 and photoperiod
control on day 2; the control group 16:8 treatment on day 3 at 3.1±0.2 x
entered the log phase on day 6. Then, the 10-2 g/L and 2.6±0.1x10-2 g/L,
treatment and control groups entered respectively (Figure 3c). Euglena sp.
the stationary phase simultaneously on in full dark treatment increased total
day 9. This meant that the photoperiod protein content from 0.24% to 0.52%,
treatment group had a longer log phase, and Euglena sp. in photoperiod 16:8
around seven days. The initial death treatment increased protein content
phase in the treatment group began on from 3.25% to 4.60%, with protein
 The effect of photoperiodism on Euglena metabolite 459

Figure 2. Growth of Euglena sp. (a) Screening potential strain of Euglena

sp.; (b) Growth of Euglena sp. in photoperiod treatment and PCA addition; (c)
Growth modelling of Euglena sp. (Full light); (d) Growth modelling of Euglena
sp. (Photoperiodism 12:12); (e) Growth modelling of Euglena sp. (Photoperiodism
14:10); (f) Growth modelling of Euglena sp. (Photoperiodism 16:8); (g) Growth
modelling of Euglena sp. (Full dark)
460 Khusnul QM, Tia E, Istini N et al.

Figure 3. Metabolite content of Euglena sp (a) carbohydrate content; (b) lipid

content; (c) protein content; (d) paramylon content; (e) pigment content.
 The effect of photoperiodism on Euglena metabolite 461

productivity of 1.1 x10-3 g/L/day and phase, namely the first and second days,
2.5 x10-3 g/L/day, respectively. Based while the control Euglena requires a
on one-way ANOVA test, the photoperiod longer time, namely the sixth day. Rapid
treatment of control Euglena sp. had exponential development in treatment
no statistically significant effect on culture begins on the second day of
carbohydrate content (p=1.310) and cultivation, reaches a stationary phase on
protein content (p=0.060). In contrast, day 9, and then begins to diminish. More
the result had a significantly different extended periods of light exposure create
effect on lipid content (p<0.05); and from photoinhibition in the cells and a lack of
the Duncan post-hoc test, there was a nutrients in the culture, causing growth
significant difference between treatment to slow down. As a result, cells could
and control groups. not complete photosynthesis to multiply
In this experiment, higher pigment (Palanisamy et al., 2022). In the control
content was obtained. The overall type treatment, the death phase began on day
of pigment chlorophyll-a and carotenoid 15. Due to decreased photosynthesis,
(Figure 3e) produced were highest in continuous light generates the lowest
the control treatment, as extensive as cell multiplication number (373±104)
1.2±00.01 x10-2 ±g/L and 0.30±0.02 and biomass productivity (0.371±21 g/L)
x10-2 g/L, respectively. The other most on day 15 of cultivation (Palanisamy et
increased production was chlorophyll-b al., 2022). Based on ANOVA test, it was
on the photoperiod treatment 14:10, as found that the photoperiod treatment did
large as 0.70±0.03x10-2 g/L. Different not significantly affect the growth rate
from pigment, higher paramylon content of Euglena. This was possibly because
was obtained using a medium with some needed light, so Euglena became
photoperiod 12:12 treatment on day more adaptive. Microalgae requires light
15 at 1.90±0.02 x10-1 g/L (Figure 3d). to produce adenosine triphosphate (ATP)
Total paramylon content of Euglena and nicotinamide adenine dinucleotide
sp. in photoperiod 12:12 treatment phosphate (NADPH2), as well as critical
increased from 45.87% to 95.47%, with chemicals for growth (Xie, Lin & Luo,
a productivity of 6.613 µg/ml/day. 2021). Besides that, light is the source
Based on the one-way ANOVA test, the of energy that drives this process, and it
photoperiod treatment of control Euglena must be considered in terms of intensity,
sp. had no statistically significant effect on spectrum quality, and photoperiod (Park
paramylon (p=0.457) and chlorophyll-b & Craggs, 2011) so that a certain level
(p=0.192) content. However, the result of intensity or quality of light may have
had a significant effect on chlorophyll a an impact on the growth rate when
content (p<0.05) and carotenoid content combined with a photoperiod.
(p<0.05); and from the Duncan post-hoc Logistic and Gompertz were two non-
test, there was a significant difference linear models that were suitable for the
between treatment and control groups rapid population growth of organisms
in chlorophyll-a and carotenoid content such as microalgae (Lam et al., 2017).
(Table 2). Not limited by substrate type and
consumption, the Logistic and Gompertz
DISCUSSION models (Figure 2c-g) were the simplest
models and could be used for general
In the initial phase of microalgae growth,
microalgae growth rate. This research
cells adjust to the new media so that
showed that the best pattern based on the
Euglena, with the addition of PCA and
Logistic and Gompertz kinetics models
photoperiod treatment, requires a lag
was on control cultivation. The resulting
462 Khusnul QM, Tia E, Istini N et al.

regression treatment was below 0.9,

0.60±0.01b

0.26±0.00a
0.30±0.00a
0.30±0.00a
0.03±0.00a
carotenoid
which deviated from the model presented.

Percent

Note: Numbers followed by a different letter indicated significant differences between treatments and were calculated by
(%)
Based on Logistic modelling, the
maximum specific growth rate (µmax)
of Euglena sp. was 0.2985/day. For the
Gompertz modelling, the maximum cell

Percent chlorophyll
production rate (rm) of Euglena sp. was

0.76±0.00

0.70±0.00
0.58±0.01
0.64±0.00
0.68±0.03
0.108 x106 cells/mL. The lag time (tL) of

b (%)
Euglena sp. was 1.428/day. Each of the
R square error value for the Logistic and
Gompertz models were 0.966 and 0.974,

Table 2. Effects of photoperiod and addition of PCA on metabolite productivity of Euglena sp.
respectively. Therefore, based on the R
square error values, the Gompertz model

Percent chlorophyll
indicated a better-fit model compared to
the Logistic model.

0.60±0.00ab
0.97±0.00b
b
1.47±0.01a

0.06±0.01c
0.21±0.00
The proportions of chemical

a (%)
components (for example, carbohydrates,
lipids, proteins, and pigments) in algal
cells are closely related to cultivation
conditions such as photoperiod and
light intensity (Juneja, Ceballos &

one-way ANOVA followed by Duncan multiple range test (DMRT) (p<0.05)

Percent para-mylon
Murthy, 2013). Proteins are the most
75.62±0.00

95.47±0.00
56.04±0.01
67.74±0.00
50.03±0.00
abundant component of dry microalgae
biomass, accounting for 6-52% of the
(%)

total; Zhu (2015) and Deng et al. (2018)

found that protein content was similar
(44.7-50.7%) when C. kessleri was
grown in a mixotrophic condition. The
highest carbohydrate production was
Percent protein

1.96±0.02

1.90±0.00
2.02±0.00
4.60±0.00
0.52±0.00
on day 9, while the most increased lipid
production was on day 12. As seen in
(%)

Figure 3, productivity of carbohydrates

on days 6, 12, and 15 were inversely
proportional to the productivity of
lipids. Carbohydrate biosynthesis is
Productivity
(g/L/day)

a competitive process in algal cells,

0.08

0.07
0.07
0.22
0.40

requiring less ATP and NADPH per carbon

than lipid synthesis (Subramanian et
al., 2013). Furthermore, microalgae
can adjust their carbon partitioning
Biomass

programmatically in response to changes

(g/L)

1.18

1.12
1.03
3.27
5.90

in culture conditions and environment

(Wang et al., 2013).
The effect of photoperiod on the
PCA-Full dark

chemical compositions of C. sorokiniana

Control (Full
Treatment

PCA-12:12
PCA 14:10

grown in CCW in a bubble-column

PCA 16:8

bioreactor were investigated. Protein

light)

content increased with increasing

illumination time, reaching a peak of
 The effect of photoperiodism on Euglena metabolite 463

54.92% at a photoperiod of 20L:4D 2020). In this research, the chlorophyll

(light:dark) (Gao et al., 2022). George content was calculated and showed
et al. (2014) also conducted similar that the control of cultivation produced
experiments on Ankistrodesmus falcatus the highest quantities of chlorophyll
and discovered that protein production and carotene pigments compared to
increased with increased illumination treatment. This indicates that continuous
duration, light intensity, and prolonged light conditions are the optimum
exposure time. As for lipid content, it conditions for producing chlorophyll
increased first, then decreased, reaching and carotenoids. However, in conditions
a maximum of 24.56% at 16:8. At of excess light, microalgae cells reduce
the same time, carbohydrate content the production and accumulation of
decreased first and then increased, chlorophyll to avoid excessive energy
reaching a minimum of 19.03% at requirements, thereby reducing
20:4. As photoperiods increased photodamage or photoinhibition due to
from 8:16 to 24:0, pigment content too much light (Li et al., 2017).
increased from 1.06 to 2.77% (Gao et Paramylon production can be
al., 2022). As previously stated, lipids increased during the exponential growth
and carbohydrates have two competing phase (Mahapatra et al., 2013), as well
pathways for storing production in as by co-culture of Pseudoalteromonas
microalgae. sp. or marine microbes during the
Short light exposure in microalgae can logarithmic phase. Another element that
form stress signals, thereby encouraging influences the existence of paramylon
cells to convert excess glucose into lipids is light circumstances which affect the
and reduce cell division (Mitra, Leeuwen accumulation time of paramylon when
& Lamsal, 2012). This stress adaptation grown in the dark versus grown in the
event is analogous to the modifications light (Zeng et al., 2016). In this research,
caused by hyperostosis or nitrogen the highest concentration of paramylon
limitation, which causes a rise in lipid obtained at treatment photoperiod
synthesis (Hirai et al., 2016). As a result, 12:12 was 95.47% for 15 days (Table
the principal chemical compositions of 2). As a result, Euglena sp. produced
algae change as light conditions change, high amounts of paramylon with a
demonstrating that photoperiod is a combination of light and darkness.
significant factor in influencing the levels As stated in a previous study, dark
of chemical compositions in algal cells. cultivation is an excellent strategy to
Cell wall resistance, solvent type, and accumulate paramylon until 80% of
extraction procedures all impact pigment total biomass (Kim et al., 2021). Besides
extraction. To extract chlorophyll that, higher irradiation levels, on the
and fucoxanthin, methanol is used other hand, contribute to carbohydrate
(Palanisamy et al., 2022). Because build up when grown in the light
chlorophyll accessory antenna transfers (Matsuda, Hayashi & Kondo, 2011). So,
photosynthetic chemical energy from 10 for the formation of paramylon, which is
NADPH2 to lipid, protein, and nucleic a carbohydrate derivative, it requires a
acid synthesis, its content must be combination of light and darkness.
quantified (Palanisamy et al., 2022). Paramylon is an insoluble, linear,
Several studies have discovered that the high-molecular-weight 1,3-glucan that
type of solvent, photoperiod, wavelength, occurs naturally in crystalline form. The
nitrogen starvation, and other physico- euglenoids accumulate paramylon in
chemical properties significantly impact granules that may be widely dispersed
pigment content (Bhattacharjya et al., throughout the cytoplasm, form caps over
464 Khusnul QM, Tia E, Istini N et al.

the pyrenoids or be packed. The various 10-2 g/L; chlorophyll-b at photoperiod

types of paramylon are classified into six 14:10 was 0.7±0.03 x10-2 g/L; and
morphological categories, all of which paramylon at photoperiod 12:12
are present in various euglenids. Uridine was 1.9±0.02x10-1 g/L. The highest
diphosphate glucose (UDP-Glc) donates carbohydrate content was found
the glucose moiety that is subsequently in control conditions, with levels of
linked to form the expanding 1.2±0.02 g/L. Euglena is a protist
polysaccharide (Skodová-Sveráková with high adaptability because it can
et al., 2020). The sugar nucleotide live as an autotroph, heterotroph,
formation depends on the enzymes UTP or mixotroph, and the presence of
(D-glucose uridylyltransferase) or UDP- a photoperiod does not significantly
Glc pyrophosphorylase (Muchut et al., influence the accumulation of Euglena
2018). In euglenoids, the appearance metabolites. Therefore, it is necessary
of paramylon requires the activity of to carry out further studies related to
paramylon synthase, a membrane- Euglena’s limitations in adapting to the
bound enzyme complex of approximately environment so that it triggers stress
670 kDa belonging to the eukaryotic and increases the targeted metabolisms.
glycosyltransferase 48 (GT48) family
(Skodová-Sveráková et al., 2020). In Acknowledgement
Euglena gracilis, the glucan synthase- This manuscript was held as a part of the first
author’s dissertation research, supported by
like genes EgGSL1 and EgGSL2 have
Pendidikan Magister menuju Doktor untuk
been identified. They encode 304 kDa Sarjana Unggul (PMDSU) Indonesia.
and 258 kDa proteins with 15 and 19
transmembrane domains, respectively Authors’ contributions
(Tanaka et al., 2017). Tanaka et Khusnul QM, principal investigator, conceptualised
al. (2017) reported that paramylon and designed the study, prepared the draft of the
manuscript and reviewed the manuscript; Tia E,
synthesis depends on the activity of advised on data analysis and interpretation, and
EgGSL2, which forms complexes with reviewed the manuscript; Istini N, led the data
37 and 54 kDa UDP-binding proteins. collection and reviewed the manuscript; Ria A,
The membrane fraction surrounding reviewed the manuscript; Dedy K, reviewed the
manuscript; Brilian RS, led the data analysis and
the paramylon granules is associated reviewed the manuscript; Revata M, conducted the
with the activity of paramylon synthase study; Bambang RA, conducted data analysis and
(Skodová- Sveráková et al., 2020). interpretation; Arief B, assisted in drafting of the
manuscript, Eko AS reviewed the manuscript.
CONCLUSION Conflict of interest
This study found that photoperiod Authors declare no conflict of interest. Authors
have received a research grant from the Indonesian
treatment had a significant effect on
Ministry of Research, Technology and Higher
lipid, chlorophyll-a, and carotenoid levels Education (Ristekdikti).
of Euglena sp. in the control treatment
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