Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73(S1): e63637, enero-diciembre 2025 (Publicado Mar. 03, 2025)

Interplay between light and temperature on growth

of Chlorella sorokiniana (Chlorellaceae) cultures

under laboratory conditions

Ana Margarita Silva Benavides1.2; https://orcid.org/0000-0001-9006-9391

Giuseppe Torzillo2,3; https://orcid.org/0000-0003-3621-8303

1. Escuela de Biología, Universidad de Costa Rica, San Pedro de Montes de Oca, San José, Costa Rica; ana.silva@ucr.ac.cr

(*Correspondence)

2. Centro de Investigación en Ciencias del Mar y Limnología, Universidad de Costa Rica, San Pedro de Montes de Oca,

San Jose, Costa Rica; giuseppe.torzillo@cnr.it

3. Consiglio Nazionale delle Ricerche-Istituto per la Bioeconomia, Via Madonna del Piano 10, Sesto Fiorentino, I-50019

Firenze, Italy.

Received 06-V-2024. Corrected 11-XI-2024. Accepted 24-I-2025.

ABSTRACT

Introduction: The relationship between light and temperature on the growth of Chlorella sorokiniana

(Chlorophyceae) cultures was investigated under laboratory conditions.

Objective: The aim of this study was to evaluate the influence of different temperature and light intensities on

growth, productivity, chlorophyll of Chlorella sorokiniana UTEX 1230 in laboratory conditions.

Methods: The cultures were exposed to a combination of two light irradiances (100 and 200 μmol photons m−2

s−1) and 5 different temperatures (20 °C, 25 °C, 30 °C, 40 °C, 45 °C).

Results: At 100 μmol photons m−2 s−1, the culture growth did not differ significantly within 20 °C and 35 °C

range. At this irradiance, the maximum attained biomass dry weight was 3.9 g/l after 9 days of cultivation under

continuous light. Cultures grown under 200 μmol photons m−2 s−1,showed much larger differences in their

growth. Their final dry weight changed according to the following temperatures, 30 °C (6.19 g/l), 25 °C (5.24

g/l), 35 °C (4.33 g/l), 40 °C (2.50 g/l) 45 °C. Therefore, the optimal temperature for productivity of cultures of

Chlorella sorokiniana, strongly changed according to the light intensities at which cultures were exposed. At 100

μmol photons m−2 s−1, a large plateau for optimal growth was observed between 20 °C and 35 °C, while at 200

μmol photons m−2 s−1 a clear optimal temperature for productivity was observed at 30 °C.

Conclusions: It was interesting to note that culture grown at 20 °C, performed well under 100 μmol photons m−2

s−1, while when exposed to 200 μmol photons m−2 s−1 they were unable to grow. No growth was achieved at 45

°C. Therefore, 40 °C represented the upper limit to appreciate the growth in Chlorella sorokiniana strain UTEX

1230, while the temperature lower limit changed with light irradiance.

Key words: Chlorella sorokiniana; growth productivity; light intensity; temperature.

RESUMEN

Interacción entre la luz y la temperatura en el crecimiento de cultivos

de Chlorella sorokiniana (Chlorellaceae) en condiciones de laboratorio

Introducción: Se investigó la relación entre la luz y la temperatura sobre la productividad de cultivos de la

microalga Chlorella sorokiniana (Chlorophyceae).

https://doi.org/10.15517/rev.biol.trop..v73iS1.63637

SUPPLEMENT

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73(S1): e63637, enero-diciembre 2025 (Publicado Mar. 03, 2025)

INTRODUCTION

Microalgae are important as primary pro-

ducers in both fresh and marine waters. They

use light energy and carbon dioxide to pro-

duce biomass. Among microalgae Chlorella

sorokiniana is a unicellular green microalga

not flagellated, spherical, 2–10 μm in diam-

eter, belonging to the phylum Chlorophyta

(Schubert, 2003). C. sorokiniana represents a

sub-species first isolated in 1953 by Sorokin,

and originally considered as a thermotolerant

mutant of Chlorella pyrenoidosa (Kunz, 1972;

Sorokin & Meyer, 1953). This taxonomic clas-

sification was thereafter revised during the

late 1980s and early 1990s by chloroplast 16S

rDNA and 18S rRNA profilin which identi-

fied C. sorokiniana as a separate species (Dorr

& Huss, 1990; Kessler & Huss, 1992). This

microalga has been proposed as source lipids

for biodiesel production, food supplements and

metabolites with pharmacological activities,

extraction of high-value compounds such as

fatty acids, pigments (carotenoids, chlorophyll)

(Becker, 2013; Borowitzka, 2013; Liu & Hu,

2013). It has also been suggested as a source of

proteins in aquafeed (Chun-Yen et al., 2023)

and as a dietary supplement meal, can have a

positive influence on the growth, antioxidant

status, and immune response of rainbow trout

main food of rotifers as live food for larvae of

marine fish (Chen et al., 2021; Lee et al., 2001)

In recent years, C. sorokiniana has been exten-

sively investigated for the wastewater treatment

coupled to the production of biodiesel (Eladel

et al., 2019).

Light and temperature are two impor-

tant fundamental environmental factors for the

autotrophic growth of microalgae since they

can affect both growth productivity and cellu-

lar biochemical composition (de la Peña, 2007;

Huesemann et al., 2018; Ugwu et al., 2007; Yang

et al., 2024. It is well known that the growth rate

of microalgae increases with the increase of

temperature and light intensity up to an opti-

mum value, and thereafter growth decreases.

Usually, the decrease in growth is much sharper

when the temperature surpasses the optimal

value than when it decreases from the optimum

(Converti et al., 2009; Ras et al., 2013).

In the literature there is enough informa-

tion on the effect of a single environmental

factor in particular temperature for C. soroki-

niana, however the information on the interac-

tion between light and temperature is scanter

(Cuaresma et al., 2012; Moronta et al., 2006;

Ugwu et al., 2007). These two environmental

factors are strategic for the outdoor cultivation

Objetivo: evaluar la influencia de diferentes temperaturas e intensidades de luz sobre el crecimiento, la producti-

vidad y la clorofila a+b de Chlorella sorokiniana UTEX 1230 en condiciones de laboratorio.

Métodos: Los cultivos se expusieron a una combinación de dos irradiaciones de luz (100 y 200 µmol fotones m-2

s-1) y cinco temperaturas (20 °C, 25 °C, 30 °C, 40 °C, 45 °C ).

Resultados: A 100 µmol fotones m-2 s-1, el crecimiento del cultivo no presentó diferencias significativas entre las

temperaturas de 20 °C y 35 °C. Con esta irradiación, el peso seco máximo de la biomasa alcanzó 3.9 g/l después

de 9 días de cultivo con luz continua. Los cultivos expuestos a una irradiación de 200 µmol fotones m-2 s-1 mos-

traron mayores diferencias en su crecimiento. El peso seco final cambió según las siguientes temperaturas, 30

°C (6.19 g/l), 25 °C (5.24 g/l), 35 °C (4.33 g/l), 40 °C (2.50 g/l), 45°C (0.00 g/l). Por consiguiente, la temperatura

óptima para la productividad de los cultivos de Chlorella sorokiniana cambió de acuerdo con las intensidades de

luz que fueron expuestos los cultivos. A 100 µmol fotones m-2 s-1 se presentó un crecimiento óptimo entre 20 °C y

35 °C, mientras que a 200 µmol fotones m-2 s-1 se observó una temperatura óptima para la productividad a 30 °C.

Conclusiones: Los resultados demuestran que los cultivos a 20 °C y 100 µmol fotones m-2 s-1 mostraron un

óptimo crecimiento; sin embargo, este fue menor cuando se expuso a 200 µmol fotones m-2 s-1. No se observó

crecimiento de los cultivos a 45 °C. Por consiguiente, la temperatura a 40°C representó el límite superior para

obtener una buena productividad en Chlorella sorokiniana cepa UTEX 1230, mientras que el límite inferior de

temperatura cambió con la irradiación de la luz.

Palabras claves: Chlorella sorokiniana; crecimiento; productividad; intensidad lumínica; temperatura.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73(S1): e63637, enero-diciembre 2025 (Publicado Mar. 03, 2025)

of microalgae, particularly light which can fluc-

tuate of one order of magnitude during the day.

The combination of low temperature and high

light conditions in the morning hours outdoors

represents a frequent situation occurring par-

ticularly in desert areas where light intensity

rises at a rate much higher than temperature

making photosynthetic apparatus prone to pho-

toinhibition with consequent reduction of daily

productivity (Torzillo et al., 1998; Vonshak et

al., 2001). C. sorokiniana, thanks to its bio-

chemical plasticity, can play an important role

in biorefinery (Sharma et al., 2022).

It is well known that the average produc-

tivity of the most common industrial strains

including C. sorokiniana is far lower than maxi-

mal theoretical estimations, suggesting that

the identification of factors limiting biomass

yield is crucial to make algal-derived bio-

products economically viable on the industrial

scale (Benedetti et al, 2018; Masojidek et al.,

2013). To acquire further information on the

growth of C. sorokiniana we exposed cells to

a combination of different temperatures rang-

ing from 20 °C to 45 °C, both at 100 and 200

µmol m-2s-1. Culture performance was followed

with both measurement of growth, and by

chlorophyll fluorescence changes to monitor

the physiological status of the cultures under

different conditions.

MATERIALS AND METHODS

Organism and culture conditions: C.

sorokiniana (Utex 1230) was obtained from the

Utex Culture Collections. The inoculum was

grown in BG-11 medium (Rippka et al., 1979)

in glass columns (5 cm internal diameter), 400

mL working volume, at 28 °C, bubbled with

a mixture of air/CO2 (97:3, v/v). The pH of

culture medium was maintained at 7.5 ± 0.1.

Cultures were illuminated with 70 μmol pho-

tons m−2 s−1 with cool white, fluorescent lights

(Dulux L, 55W/840, Osram, Italy).

Experimental conditions: C. sorokiniana

was grown in a batch mode. All experiments

were conducted using triplicate 400 ml glass

columns. Experiments were carried out under

continuous illumination of 100 and 200 μmol

photons m−2 s−1, and at 6 different temperatures

(20, 25, 30, 35, 40 and 45 °C). The photon flux

density (PFD) was measured on the surface

of tubes using the LI-250A equipped with a

flat quantum sensor (LI-COR Biosciences, NE,

USA). Cultures were grown under continuous

illumination (24/00). Each tube was bubbled

with a sterilized mixture of air/CO2 (97: 3, v/v)

at the rate of 51 min-1 to maintain turbulence

and to keep the pH between 7.0 and 7.5. The

flask cultures were inoculated with an initial

dry weight of about 100 mgL-1. Both culture

medium and glass columns were autoclaved for

45 min at 121 °C to prevent any contamination

during the growth experiments.

Dry weight: Dry weight was performed

on triplicate 10 mL samples. Samples were

taken at 24 hours intervals to determine the

cell dry weight increase. Each sample was

filtered through 0.7 μm pore size pre-weight-

ed membranes (Whatman grade GF/F filters,

Maidstone, England) and then dried at 105 °C

for 3 h. They were then transferred in a desic-

cator to equilibrate them to room temperature,

and thereafter the filters were weighed. Bio-

mass productivity (mg l-1 d-1) was calculated

according to the equation x1-x0 /∆t where x1

and x0 (g/l) were the dry weight at intervals of

24 hours (∆t).

Pigments: To measure cell chlorophyll a/b,

and total carotenoids contents, a 10 ml aliquot

of each culture was centrifuged for 5 min at

4000 x g. Five ml of 90 % acetone (v/v) were

then added to the pellet, mixed in the vortex for

5 min, centrifuged and determined spectropho-

tometrically according to Lichtenthaler (1987)

using a Beckman DU-640 spectrophotometer.

Chlorophyll fluorescence: Daily measure-

ments of the fluorescence parameter Fv/Fm,

i.e., the maximum photochemical quantum

yield of PSII, were performed with porta-

ble pulse-amplitude-modulation fluorometer

(PAM 2500, H. Walz, Effeltrich, Germany). For

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73(S1): e63637, enero-diciembre 2025 (Publicado Mar. 03, 2025)

this purpose, a volume of 1.5 ml of algal cul-

ture taken directly from the culture tubes, was

incubated in the dark for 10–15 min in a 2 ml

culture volume cuvette. PSII minimum fluores-

cence (F0) was measured with a low-intensity

modulated measuring beam (0.3 μmol m-2 s-1)

from light-emitting diodes (peak wavelength at

650 nm, frequency 600 Hz). A saturating light

pulse was then administered to reach Fm level

(Masojidek et al., 2011; Maxwell & Johnson,

2000; Torzillo et al., 1998).

Statistical analysis: Data gathered from

laboratory batch cultures were treated statisti-

cally by two-way analysis of variance (ANOVA).

Bonferroni’s post-test was performed to deter-

mine the statistical significance (P < 0.05) of the

effects of light and temperature and interaction

of both factors on growth, productivity, chlo-

rophyll a+b. The statistical analysis was carried

out using GraphPad Prism 5 (GraphPad Soft-

ware, Inc., California, USA).

RESULTS

In Fig. 1, the growth curves of C. soroki-

niana cells grown at different temperatures

ranging from 20 °C to 45 °C and at two light

irradiances, 100 μmol photons m−2 s−1 (Fig.

1A) and 200 μmol photons m−2 s−1 are shown.

(Fig. 1B).

Maximum dry weight reached by the cul-

tures was about double in the cultures grown

at double irradiance (100 vs 200 μmol photons

m-2s-1). However, the optimal temperature for

growth showed a broad range in cultures grown

under 100 μmol photons m-2s-1), spanning

from 20 °C to 35 °C, and peaking at 25 °C, while

in cultures grown under 200 μmol photons

m-2s-1, the optimal growth was found at 30 °C

(Fig. 1B) (P < 0.001). An interesting feature

was the different lower temperature thresholds

for growth. Cultures grew well at 20 °C, under

100 μmol photons m-2s-1, while no appreciable

growth was obtained at the same temperature

but with double irradiance (P < 0.001). When

the culture temperature was raised to 45 °C no

appreciable growth was achieved under both

the irradiances (Fig. 2A, Fig. 2B) (P > 0.05).

Productivities achieved at different growth

temperatures increased with the irradiance from

100 μmol photons m-2s-1 (Fig. 2A) to 200 μmol

photons m-2s-1 (Fig. 2B) (P < 0.001). Interest-

ingly, the optimal temperature for growth was

recorded at 25 °C at the lower light irradiance

(Fig. 2A), and at 30 °C at higher irradiance (Fig.

2B), that is, there was a shift of 5 °C in the opti-

mal temperature for growth between the two

tested irradiances. Moreover, under 100 μmol

photons m-2s-1 (Fig. 2A) the differences in pro-

ductivities within the range of temperature of

20 °C and 35 °C, although significant, (P < 0.05)

were not as high as that found under 200 μmol

photons m-2s-1 In addition, under the higher

irradiance (Fig. 2B) the decline in productivity

was more marked than that found with a lower

irradiance (Fig. 2A) as the temperature moved

away from the optimum. Surprisingly, at 20 °C

and 200 μmol photons m-2s-1 the productiv-

ity was negligible (Fig. 2B), most likely as a

Fig. 1. Dry weight increase attained at different temperatures by C. sorokiniana UTEX 1230. A. PFD = 100 μmol photons

m-2s-1, B. PFD= 200 μmol photons m-2s-1

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73(S1): e63637, enero-diciembre 2025 (Publicado Mar. 03, 2025)

result of high irradiance combined to low tem-

perature (P < 0.001). Both at 100 and 200 μmol

photons m-2s-1, no productivity was detected at

45 °C indicating that 40 °C is the upper limit for

growth productivity (P > 0.05).

Chlorophyll fluorescence: In Fig. 3, the

changes in the Fv/Fm ratio of the cultures

grown at different temperatures and under the

two irradiances are shown. The Fv/Fm ratio of

culture grown at 100 μmol photons m−2 s−1

remained close to the optimal value (0.7 ±

0.05) within the temperatures range 20–40 °C,

with a tendency to diminish as the temperature

increased. The highest value 0.744 (P < 0.05)

was attained by the cultures grown at 25 °C and

100 μmol photons m−2 s−1.

However, when cultures were exposed to

45 °C, the decline of F/Fm value become evi-

dent indicating the inhibition of the photo-

synthetic activity, in line to what observed by

growth measurements (Fig. 3A). The Fv/Fm

ratio measured during the 8 consecutive days

of culture grown under 200 μmol photons m−2

s−1 resulted generally inferior to that of culture

grown at 100 μmol photons m−2 s−1(P < 0.001).

Under 200 µmol photons m-2s-1 the lowest

values were observed at 45 °C, 0.11 (average

of 8 days). Fv/Fm values significantly lower

than optimal were recorded at 20 °C, particu-

larly under 200 μmol photons m−2 s−1: at this

temperature, Fv/Fm was 0.382 (average) with

tendency to reduction during the cultivation

period. Therefore, the combination of subop-

timal temperature and higher light caused a

negative synergistic effect on the maximum

photochemical quantum yield of PSII, at both

20 °C (Fig. 3).

Chlorophyll: Total cellular chlorophyll

was strongly modified by both light intensity

and temperature (Fig. 4A, Fig. 4B) (P < 0.001).

In general, the amount of chlorophyll accumu-

lated by the cells resulted higher in cultures

grown under 100 μmol photons m-2s-1 (Fig.

4A). Under this light irradiance, the highest

accumulation was observed within 20 °C and

35 °C, with a significant difference (P < 0.05)

in favor of the culture grown at 30 °C (Fig. 4A).

A 40 °C chlorophyll accumulation showed a

Fig. 2. Productivity (mgL-1day-1) of C. sorokiniana UTEX 1 230 cultures grown at different temperatures and light intensities,

A. PFD = 100 and B. PFD = 200 μmol photons m-2s-1. No appreciable productivity was achieved at 45 °C both at 100, and

200 μmol photons m-2s-1, and at 20 °C under 200 μmol photons m-2s-1.

Fig. 3. Maximum photochemical efficiency of PSII (Fv/Fm),

of C. sorokiniana Utex 1230 cells grown at both 100 and 200

μmol photons m−2 s−1 and different temperatures.

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73(S1): e63637, enero-diciembre 2025 (Publicado Mar. 03, 2025)

strong drop, and no new synthesis was attained

at 45 °C. (Fig. 4A). In cultures grown under

200 μmol photons m-2s-1, no significant differ-

ences were observed within 25 °C and 35 °C

(P > 0.05), while at 40 °C chlorophyll showed

a strong reduction, and a further rise of tem-

perature to 45 °C caused a total inhibition of

chlorophyll synthesis (Fig. 4B). The exposure

of cultures to double level of irradiance at 20 °C

caused an inhibition of synthesis of chlorophyll

(Fig. 4B). Data gathered with chlorophyll mea-

surements align with those of growth. Indeed,

no growth was attained at 45 °C under both the

light irradiances and similarly no growth, and

thus no productivity could be detected at 20 °C

under double the amount of light (Fig. 4B).

Total carotenoids: Total carotenoids of the

cells varied under both light regimes and at var-

ious temperatures. The data reported in Fig. 5

show that a higher content of total carotenoid

was recorded at a lower irradiance (P < 0.001).

However, a different pattern was observed

with temperature. Under 100 μmol photons

m-2s-1, the amount of carotenoids decreased

linearly to zero when the growth temperature

was increased to 45 °C. Under 200 μmol m-2s-

1, the higher carotenoid content was attained

by cultures grown within the 25 °C and 40 °C

range, while no significant amounts of carot-

enoids were recorded at both 20 °C and 45 °C.

(Fig. 5).

DISCUSSION

Microalgal grown outdoors are subjected

to suboptimal culture conditions, particularly

light and temperature, two environmental fac-

tors that are difficult to govern even through the

adoption of an appropriate culture system. Cul-

tures grown in open ponds usually suffer from

low temperatures, particularly in the morning

hours, while in closed systems they are usually

subjected to excessive temperature. Microal-

gal cultures, grown both in open and closed

systems, suffer from excessive light irradiance

during the central hour of the day (Torzillo

& Vonshak, 2013). Therefore, an important

aspect of algal biotechnology is to develop

strains that can grow well both under subop-

timal and supra-optimal temperatures, that is,

within 20 °C and 40 °C. Culture temperatures

close to 40 °C can be frequently recorded even

in open ponds, particularly in greenhouses in

Fig. 4. Total chlorophyll (a+b) accumulations attained in culture of C. sorokiniana exposed at two light irradiances,100 μmol

photons m-2s-1 A., and of 200 μmol photons m-2s-1, B. at different temperatures.

Fig. 5. Total carotenoid content detected in culture of C.

sorokiniana exposed to two light irradiances (100 and 200

μmol photons m-2s-1) and temperatures from 20 to 45 °C.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73(S1): e63637, enero-diciembre 2025 (Publicado Mar. 03, 2025)

summer (Torzillo et al., 2021), while 20 °C and

lower values are usually recorded in the early

morning hours and over the day at the end of

culture season. However, it is important to con-

sider that cultures outdoors are subjected to the

interaction of two or more environmental fac-

tors, usually light and temperature. Therefore,

the development of strains that are capable of

growing under variable light and tempera-

ture conditions, and that preserve a standard

biochemical composition, as usually required

by the market, are important prerequisites to

consider to properly address the selection of

suitable strains (Seyfabadi et al., 2011). In this

respect, C. sorokiniana has evidenced a broad

range of temperatures for growth, particularly

under lower irradiance ranging from 20 °C to

40 °C. Interestingly, the productivity reached

at 20 °C under low irradiance was comparable

to that reached at 25 °C (optimal). This is an

important feature of the strain for growing it

in temperate regions. Actually, this strain is

successfully grown in thin-layer cascades in

Central Europe (Grivalsky et al., 2019). More-

over, the ability to resist until 40 °C adds further

advantages in the choice of this strain outdoors.

It was interesting to note that optimal

productivity was shifted by 5 °C, that is, from

25 °C to 30 °C when the light irradiance was

increased to 200 µmol m-2-s-1 evidencing the

synergistic effect of light and temperature. This

finding confirmed that the selection of strains

must include a combination of more than one

factor to better evaluate their performance.

Another interesting finding was that growth

of the cultures at 20 °C under lower light reached

productivity comparable to that attained at 25

°C, and this was probably achieved by shifting

the cellular metabolism toward the production

of carbohydrates that were used as a sink to

store the excess of reducing power (Torzillo et

al., 1991). Surprisingly, no growth was achieved

at 20 °C when cultures were grown under 200

µmol m-2-s-1. The measurement of chlorophyll

fluorescence clearly indicated a strong depres-

sion of the Fv/Fm ratio indicating that cultures

were photo inhibited. Indeed, Fv/Fm which is an

indicator of photoinhibition (Masojidek et al.,

2011; Torzillo et al., 1998; Vonshak et al., 1994)

was very low, about 0.4, under the combination

of light and low temperature. Conditions of

suboptimal temperature and high light in low

light acclimated cultures, as those growing out-

doors, is a situation that can frequently occur

in the morning hours where the rise of light is

much faster than that of temperature creating

the conditions of photoinhibition at relatively

low light (Vonshak et al., 2001). Indeed, low

temperatures in low acclimated cultures can

make the photosynthetic apparatus prone to

photoinhibition even in relatively low light

irradiance due to an over reduction of the pho-

tosynthetic carriers (Torzillo & Vonshak, 2013;

Vonshak et al., 2001).

Generally, the average productivity of the

most common industrial strains, including C.

sorokiniana, is much lower than the theoretical

maximum, therefore it is mandatory to iden-

tify and assess the incidence of factors limit-

ing productivity. According to Huesemann et

al. (2018) working with C. sorokiniana, a key

challenge in the development of an economi-

cally viable biofuels production process was the

identification of strains that exhibit an annual

biomass productivity of at least 25 g m−2 day−1

which is about double that achieved with pres-

ent know-how on algal biotechnology.

The information gathered with this study

on the interplay of environmental factors on the

growth of C. sorokiniana point out the necessity

to select robust strains resilient to changes in

temperature and light irradiance. This spe-

cies was able to growth within a wide range

of temperatures, ranging from 20 °C to 40 °C,

when exposed to low irradiance. Therefore, it

is advisable for mass culture of this species, the

choice of photobioreactor designs in which it is

possible to achieve a high dilution rate of light

irradiance, that is, vertical plates placed close to

each other, or compact vertical tubular reactors

to increase the cross-section of the reactors and

diminish the incident light irradiance imping-

ing on the surface of the reactor (Cuaresma

et al., 2009; Slegers et al., 2011; Torzillo et al.,

1991; Torzillo et al., 2022; Tredici et al., 2015).

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73(S1): e63637, enero-diciembre 2025 (Publicado Mar. 03, 2025)

In conclusion, the present findings dem-

onstrated that C. sorokiniana Utex 1230 rather

than to be a thermotolerant strain, by converse

it can be grown under the suboptimal tem-

perature of 20 °C without a remarkable loss

in productivity, making it attractive for cul-

tivating under temperate European regions

where mesophilic commercial strains, such as

Arthrospira platensis, are difficult to cultivate

for extended.

Ethical statement: the authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are fully

and clearly stated in the acknowledgments sec-

tion. A signed document has been filed in the

journal archives.

ACKNOWLEDGMENTS

We thank to Consiglio Nazionale delle

Ricerche-Istituto per la Bioeconomia, Via

Madonna del Piano 10, Sesto Fiorentino, I-50019

Florence, Italy for the laboratory support.

REFERENCES

Becker, W. (2013). Microalgae for human and animal nutri-

tion. In A. Richmond & Q. Hu, (Eds.), Handbook of

microalgal culture: applied phycology and biotechnolo-

gy (pp. 461–503). John Wiley y Sons.

Benedetti, M., Vecchi, V., Barera, S., & Dall’Osto, L. (2018).

Biomass from microalgae: the potential of domes-

tication towards sustainable biofactories. Micro-

bial Cell Factories, 17, 173. https://doi.org/10.1186/

s12934-018-1019-3

Borowitzka, M. (2013). High-value products from microal-

gae-their development and commercialization. Jour-

nal of Applied Phycology, 25, 743–756.

Chen, W., Luo, L., Han, D., Long, F., Chi, Q., & Hu, Q.

(2021). Effect of dietary supplementation with Chlo-

rella sorokiniana meal on the growth performance,

antioxidant status, and immune response of rain-

bow trout (Oncorhynchus mykiss). Journal of Applied

Phycology, 33, 3113–3122. https://doi.org/10.1007/

s10811-021-02541-w

Chun-Yen, C., Jhih-Ci, L., Yu-Han, C., Jih-Heng, C.,

Dillirani, N., Duu-Jong, L., & Jo-Shu, C. (2023).

Optimizing heterotrophic production of Chlorella

sorokiniana SU-9 proteins potentially used as a sus-

tainable protein substitute in Aquafeed. Bioresource

Technology, 370, 128538. https://doi.org/10.1016/j.

biortech.2022.128538

Converti A., Casazza, A. A., Ortiz, E. Y., Perego, P., &

Borghi, M.. (2009). Effect of temperature and nitro-

gen concentration on the growth and lipid content

of Nannochloropsis oculata and Chlorella vulgaris

for biodiesel production. Chemical Engineering and

Processing, 48, 1146–1151. https://doi.org/10.1016/j.

cep.2009.03.006

Cuaresma, M., Janssen, M., Vílchez, C., & Wijffels, R.

(2009). Productivity of Chlorella sorokiniana in a

short light-path (SLP) panel photobioreactor under

high irradiance. Biotechnology and Bioengineering,

104(2), 352–359. https://doi.org/10.1002/bit.22394

Cuaresma, M. F., Buffing, M. F., Janssen, M., Vílchez

Lobato, C., & Wijffels, R. H. (2012). Performance of

Chlorella sorokiniana under simulated extreme win-

ter conditions. Journal of Applied Phycology, 24(4),

693–699. https://doi.org/10.1007/s10811-011-9687-y

De La Peña, M. R. (2007). Cell growth and nutritive value of

the tropical benthic diatom, Amphora sp., at varying

levels of nutrients and light intensity, and different

culture locations. Journal of Applied Phycology, 19,

647–655. https://doi.org/10.1007/s10811-007-9189-0

Dorr, R., & Huss, V. A. R. (1990). Characteriza-

tion of nuclear-DNA in 12 species of Chlore-

lla (Chlorococcales, Chlorophyta) by DNA

reassociation. Biosystems, 24(2), 145–155. https://doi.

org/10.1016/0303-2647(90)90007-N

Eladel, H., Abomohra, A. E. F., Battah, M., Mohmmed, S.,

Radwan, A., & Abdelrahim, H. (2019). Evaluation of

Chlorella sorokiniana isolated from local municipal

wastewater for dual application in nutrient removal

and biodiesel production. Bioprocess and Biosystems

Engineering, 42, 425–433. https://doi.org/10.1007/

s00449-018-2046-5

Grivalsky, T., Ranglova, K., Câmara Manoel, J. A., Lakatos,

G. E., Lhotsky, R., & Masojidek, J. (2019). Develop-

ment of thin-lyer cascades for microalgae cultivation:

milestones (review). Folia Microbiologica, 64, 603–

614. https://doi.org/10.1007/s12223-019-00739-7

Huesemann, M., Chavis, A., Edmundson S., Rye, D.,

Hobbs, S., Sun, N., & Wigmosta, M. (2018). Climate-

simulated raceway pond culturing: quantifying the

maximum achievable annual biomass productivity

of Chlorella sorokiniana in the contiguous USA.

Journal Applied Phycology, 30, 287–298. https://doi.

org/10.1007/s10811-017-1256-6

Kessler, E., & Huss, V. A. R. (1992). Comparative physio-

logy and biochemistry and taxonomic assignment

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73(S1): e63637, enero-diciembre 2025 (Publicado Mar. 03, 2025)

of the Chlorella (Chlorophyceae) strains of the cul-

ture collection of the University of Texas at Austin.

Journal of Phycology, 28(4), 550–553. https://doi.

org/10.1111/j.0022-3646.1992.00550.x

Kunz, W. F. (1972). Response of the alga Chlorella sorokinia-

na to 60 Co gamma radiation. Nature, 236, 178–179.

https://doi.org/10.1038/236178a0

Lee, J. S., Kim, D. K., Lee, J. P., Park, S. C., Koh, J. H., & Ohh,

S. J. (2001). CO2 fixation by Chlorella KR-1 using flue

gas and its utilization as a feedstuff for chicks. Journal

of Microbiology and Biotechnology, 11(5), 772–775.

Lichtenthaler, H. (1987). Chlorophyll and carotenoids:

pigments of photosynthetic membranes. Methods of

Enzymology, 148, 350–382.

Liu, J., & Hu, Q. (2013). Chlorella: industrial production

of cell mass and chemicals. In A. Richmond, & Q.

Hu (Eds.), Handbook of microalgal culture: applied

phycology and biotechnology (pp. 329–338). John

Wiley y Sons.

Masojídek, J., Torzillo, G., & Koblízek, M. (2013). Pho-

tosynthesis in microalgae. In A. Richmond, & Q.

Hu (Eds.), Handbook of microalgal culture: applied

phycology and biotechnology (pp. 21–36). John Wiley

y Sons.

Masojidek J., Vonshak, A., & Torzillo, G. (2011). Chloro-

phyll fluorescence applications in microalgal mass

cultures. In D. J. Suggett, O. Prašil & M. A. Borowitzka

(Eds.), Chlorophyll a fluorescence in aquatic sciences:

methods and applications developments in applied

phycology (pp. 277–292). Springer Science+Business

Media B.V.

Maxwell, K., & Johnson, G. N. (2000). Chlorophyll fluo-

rescence- a practical guide. Journal of Experimental

Botany, 51(345), 659–668. https://doi.org/10.1093/

jexbot/51.345.659

Moronta, R., Mora, R., & Morales, E. (2006). Response of

the microalga Chlorella sorokiniana to pH, salinity

and temperature in axenic and non axenic conditions.

Revista de la Facultad de Agronomía, 23, 27–41.

Ras, M., Steyer, J. P., & Bernard, O. (2013). Temperature

effect on microalgae: A crucial factor for outdoor

production. Reviews in Environmental Science and

Biotechnology, 12, 153–164. https://doi.org/10.1007/

s11157-013-9310-6

Rippka, R., Deruelles, J., Waterbury, B., Herdman, M., &

Stanier, R. Y. (1979). Generic assignments, strain

histories and properties of pure cultures of Cyanobac-

teria. Journal of General Microbiology, 111(1), 1–61.

https://doi.org/10.1099/00221287-111-1-1

Seyfabadi, J., Ramezanpour, Z., & Amini-Khoeyi, Z.

(2011). Protein, fatty acid, and pigment content

of Chlorella vulgaris under different light regimes.

Journal of Applied Phycology, 23, 721–726. https://doi.

org/10.1007/s10811-010-9569-8

Sharma, P., Srinivas Gujjala, L. K., Varjani, S., & Kumar, S.

(2022). Emerging microalgae-based technologies in

biorefinery and risk assessment issues: bioeconomy

for sustainable development. Science of the Total

Environoment, 813, 152417. https://doi.org/10.1016/j.

scitotenv.2021.152417

Schubert, E. (2003). Nonmotile coccoid and colonial green

algae. In J. D. Wehr, & R. Sheath (Eds.), Freshwater

algae of North America: ecology and classification

(2nd ed., pp. 253–307). Academic Press. https://doi.

org/10.1016/B978-0-12-385876-4.00007-4

Sleger, P. M., Wijffels, R. H., van Straten, G., & van Boxtel,

A. J. B. (2011). Design scenarios for flat panel pho-

tobioreactors. Applied Energy, 88(10), 3342–3353.

https://doi.org/10.1016/j.apenergy.2010.12.037

Sorokin, C., & Myers, J. (1953). A high-temperature strain

of Chlorella. Science, 117(3039), 330–331. https://doi.

org/10.1126/science.117.3039.330

Torzillo, G., Bernardini, P., & Masojidek, J. (1998). Online

monitoring of chlorophyll fluorescence to assess the

extent of photoinhibition of photosynthesis induced

by high oxygen concentration and low tempera-

ture and its effect on the productivity of outdoor

cultures of Spirulina platensis (Cyanobacteria).

Journal of Phycology, 34(3), 504–510. https://doi.

org/10.1046/j.1529-8817.1998.340504.x

Torzillo, G., Chini Zittelli, G., Silva Benavides, A. M.,

Ranglova, K., & Masojidek, J. (2021). Culturing of

microalgae for food applications. In T. Lafarga &

G. Acién (Eds.), Cultured Microalgae for the Food

Industry (pp. 1–48). Academic Press. https://doi.

org/10.1016/B978-0-12-821080-2.00002-2

Torzillo, G., Sacchi, A., & Materassi, R. (1991). Tempe-

rature as an important factor affecting producti-

vity and night biomass loss in Spirulina platensis

grown outdoors in tubular photobioreactors. Bio-

resource Technology, 38(2–3), 95–100. https://doi.

org/10.1016/0960-8524(91)90137-9

Torzillo, G., & Vonshak, A. (2013). Environmental stress

physiology with references to mass cultures. In A.

Richmond (Ed.), Handbook of microalgal mass cultu-

res (pp. 90–113). Blackwell Science.

Torzillo G., Zittelli, G. C., Cicchi, B., Diano, M., Parente, M.,

Silva-Benavides, A. M., Esposito, E., & Touloupakis,

E. (2022). Effect of plate distance on light conversion

efficiency of a Synechocystis culture grown outdoors

in a multiplate photobioreactor. Science of the Total

Environment, 842, 156840. https://doi.org/10.1016/j.

scitotenv.2022.156840

Tredici, M. R., Bassi, N., Prussi, M., Biondi, N., Rodolfi,

L., Chini Zittelli, G., & Sampietro, G. (2015). Energy

balance of algal biomass production in a 1-ha “Green

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73(S1): e63637, enero-diciembre 2025 (Publicado Mar. 03, 2025)

Wall panel” plant: how to produce algal biomass

in a closed reactor achieving a high net energy

ratio. Applied Energy, 154, 1103–1111. https://doi.

org/10.1016/j.apenergy.2015.01.086

Ugwu C. U., Aoyagi H., & Uchiyama H. (2007). Influence of

irradiance, dissolved oxygen concentration, and tem-

perature on the growth of Chlorella sorokiniana. Pho-

tosynthetica, 45(2), 309–311. https://doi.org/10.1007/

s11099-007-0052-y

Vonshak, A., Torzillo, G., Masojidek, J., & Boussiba, S.

(2001). Sub-optimal morning temperature induces

photoinhibition in dense outdoor cultures of the

alga Monodus subterraneus (Eustigmatophyta). Plant

Cell Environment, 24(10), 1113–1118. https://doi.

org/10.1046/j.0016-8025.2001.00759.x

Vonshak, A., Torzillo, G., & Tomaselli, L. (1994). Use of

chlorophyll fluorescence to estimate the effect of pho-

toinhibition in outdoor cultures of Spirulina platensis.

Journal of Applied Phycology, 6, 31–34. https://doi.

org/10.1007/BF02185901

Yang, J., Dou, S., Liu, X., Zhu, L., Liu, K., Zhang, Y., Li, L.,

Liu, G., & Yang, M. (2024). Enhanced starch accumu-

lation in Chlorella sorokiniana as sugar platform and

the expression profiling of key regulatory proteins.

Industrial Crops & Products, 213, 118433. https://doi.

org/10.1016/j.indcrop.2024.118433