PRODUCTION OF BIOMASS AND LIPID YIELDS OF
CHLORELLA SP. CULTIVATED IN AUTOTROPHIC AND
MIXOTROPHIC MEDIA
PRODUCCIÓN DE BIOMASA Y RENDIMIENTOS
LÍPIDOS DE CHLORELLA SP. CULTIVADO EN MEDIOS
AUTÓTROFOS Y MIXOTRÓFICOS
Karla Eugenia Díaz Santiago
Universidad de Ciencias y Artes de Chiapas, México
Pascual López de Paz
Universidad de Ciencias y Artes de Chiapas, México
Arnulfo Rosales Quintero
Instituto Tecnológico de Tuxtla, México
Paris Hiram Espinosa López
Universidad de Ciencias y Artes de Chiapas, México
Paula Deyanira Orantes Calleja
Tecnológico Nacional de México
Héber Vilchis Bravo
Universidad de Ciencias y Artes de Chiapas, México
Jorge Evaristo Conde Díaz
Universidad de Ciencias y Artes de Chiapas, México
pág. 4533
DOI: https://doi.org/10.37811/cl_rcm.v8i5.13912
Production of Biomass and Lipid Yields of Chlorella sp. Cultivated in
Autotrophic and Mixotrophic Media
Karla Eugenia Díaz Santiago
1
Diaz.keugenia@gmail.com
https://orcid.org/0000-0002-9513-468X
Instituto de Investigación e Innovación
en Energías Renovables
Universidad de Ciencias y Artes de Chiapas
Tuxtla Gutierrez, Chiapas
Pascual López de Paz
pascual.lopez@unicach.mx
https://orcid.org/0000-0003-1120-3834
Instituto de Investigación e Innovación
en Energías Renovables
Universidad de Ciencias y Artes de Chiapas
Tuxtla Gutierrez, Chiapas
Arnulfo Rosales Quintero
arquinte@tuxtla.tecnm.mx
https://orcid.org/0000-0001-5609-7532
Instituto Tecnológico de Tuxtla
Tuxtla Gutiérrez, Chiapas
Paris Hiram Espinosa López
p.espinosa.idea@gmail.com
https://orcid.org/0000-0002-7956-1438
Instituto de Investigación e Innovación
en Energías Renovables
Universidad de Ciencias y Artes de Chiapas
Tuxtla Gutierrez, Chiapas
Paula Deyanira Orantes Calleja
pauuorc@gmail.com
https://orcid.org/0000-0002-9160-7005
División de Ingeniería en Energías Renovables
Tecnológico Nacional de México
Tecnológico de Estudios Superiores
de San Felipe del Progreso
Estado de México
Héber Vilchis Bravo
heber.vilchis@unicach.mx
https://orcid.org/0000-0001-6042-9851
Instituto de Investigación e Innovación
en Energías Renovables
Universidad de Ciencias y Artes de Chiapas
Tuxtla Gutierrez, Chiapas
Jorge Evaristo Conde Díaz
jconded@yahoo.com.mx
https://orcid.org/0000-0002-7992-2950
CONAHCYT
Universidad de Ciencias y Artes de Chiapas
Instituto de Investigación e Innovación en
Enegías Renovables
Tuxtla Gutiérrez, Chiapas
1
Autor principal
Correspondencia: Diaz.keugenia@gmail.com
pág. 4534
ABSTRACT
In this work, we analyze the biomass and lipid yields of Chlorella sp., when it grows in synthetic
wastewater with and without nitrogen limitation and in both cases, adding glucose to both medium,
autotrophic and mixotrophic. Our results confirm that it is possible to expand their possibilities of use,
which range from their use for the bioremediation of bodies of water to obtaining various biofuels due
to their high content of lipids and carbohydrates. It was identified that both the biomass and lipids
were higher in the media with mixotrophy with 535.71 mg L
-1
and 244.60 mg L
-1
, respectively.
Similarly, the importance of nitrogen present in the growth medium was recognized as a determining
variable for the accumulation of lipids in the species, while it is concluded that the use of Chlorella
sp. eliminates a significant percentage of nitrogen and phosphorus present in wastewater, thereby
reducing nutrient contamination. The nutrient stress to which the microalgae were subjected allowed a
greater accumulation of lipids in the cells, which leads to the conclusion that in a large-scale study,
Chlorella sp. It could be used as a raw material to obtain oils and their subsequent transformation into
biodiesel.
Keywords: chlorella, biomass, lipid yields, bioremediation, mixotrophy medium
pág. 4535
Producción de Biomasa y Rendimientos Lípidos de Chlorella sp. Cultivado
en Medios Autótrofos y Mixotróficos
RESUMEN
En este trabajo, analizamos los rendimientos de biomasa y lípidos de Chlorella sp., cuando crece en
aguas residuales sintéticas con y sin limitación de nitrógeno y en ambos casos, adicionando glucosa al
medio, tanto autótrofo como mixotrófico. Nuestros resultados confirman que es posible ampliar sus
posibilidades de uso, que van desde la biorremediación de cuerpos de agua hasta la obtención de
diversos biocombustibles por su alto contenido en lípidos y carbohidratos. Se identificó que tanto la
biomasa como los lípidos fueron mayores en los medios mixotroficos con 535,71 mg L
-1
y 244,60 mg
L
-1
, respectivamente. De igual forma, se determinó la importancia del nitrógeno presente en el medio
de crecimiento como variable determinante para la acumulación de lípidos en la especie, mientras que
se concluye que el uso de Chlorella sp. elimina un importante porcentaje de nitrógeno y fósforo
presentes en aguas residuals; reduciendo así la contaminación por nutrientes. El estrés nutritivo al que
fueron sometidas las microalgas permitió una mayor acumulación de lípidos en las células, lo que
lleva a concluir que en un estudio a gran escala, Chlorella sp. podría utilizarse como materia prima
para la obtención de aceites y su posterior transformación en biodiesel.
Palabras clave: chlorella, biomasa, rendimiento de lípidos, bioremediación, medio mixotrofico
Artículo recibido 08 setiembre 2024
Aceptado para publicación: 30 setiembre 2024
pág. 4536
INTRODUCTION
Microalgae represent a promising raw material not only in the food, pharmaceutical and cosmetic
industries [1] but also due to their capacity to absorb CO
2
within photosynthetic their process, and
their manipulable composition of lipids, proteins or carbohydrates, which can be used to obtain
biofuels or bioproducts [2].
Its promising use as clean technology lies in the fact that, in any growing condition, it yields high
population rates after just a few hours, compared to any terrestrial plant [3].
To produce microalgae, there are different forms of cultures; in any of them, it is necessary to use a
device called photobioreactors, which allow the conversion of light and carbon mainly into biomass
[4]. An adequate design of these photobioreactors involves considering the environmental conditions
of the place where the microalgae are to be grown and characteristics of the type of microalgae that
will be worked with [5].
However, the main challenge of microalgae technology lies in improving biomass production and
being able to obtain high lipid accumulation for its subsequent transformation into value-added
products, all this on a commercial scale and using only one type of culture [6]. These limitations come
mainly from the low photosynthetic efficiency achieved by currently used devices and the cost of
adding an organic or inorganic carbon source to the process [7, 8]. A viable option for increase the
growth rate in species such as Chlorella sp. is the exploration of their metabolic pathway in different
types of cultures [6].
Although autotrophic cultures would use CO
2
from the environment, it would be difficult to maintain
a high cell density due to the variability in light penetration of the entire culture [7]. In contrast, while
heterotrophic cultures would efficiently convert the organic carbon present, maintenance costs would
be a negative factor [8].
Thus, in recent years, research has opted for the making of mixotrophic cultures, where there is an
organic carbon source (such as glycerol, glucose, or acetate) and an inorganic one simultaneously
while in the presence of light [9]. As a result, increased biomass productions are obtained, up to three
times higher than autotrophic cultures [2].
pág. 4537
In this type of culture, careful handling must be taken with the concentration of essential nutrients
present in the medium, like carbon, nitrogen, and phosphorus; since their limitation or enrichment
could trigger microalgal inhibition [10]. Research shows that nitrogen limitation stress favors the
accumulation of lipids in cells; however, it decreases biomass production, which continues to
represent a bottleneck production [11]. On the other hand, the addition of glucose represents a carbon
source that allows increasing the growth rate of such microorganisms, some species of Chlorella have
reported a higher biomassic and lipidic yield in mixotrophic cultures where the concentrations of
carbon and nitrogen have been variable. [12].
It is possible to obtain a value-added product in the production of biofuels with microalgae; the
culture medium can be wastewater from urban sources due to its high nitrogen and phosphorus
content [13]. Which, once assimilated by the microorganisms, would represent a decrease in the
eutrophication of waste water bodies [14].
For all the above, the present work evaluated Chlorella sp.´s growth and lipid content using synthetic
wastewater with and without nutrient limitation, adding a source of organic carbon to the culture
medium.
METHODOLOGY
Microalgae Culture
Chlorella sp. was cultured in synthetic medium BG11 [15] in 250 mL flasks, at 24 ± 2°C, for 15 days.
With an artificial light source of 3000 lux of luminance at a 12:12 cycle photoperiod. Without the
addition of external CO
2
.
Growth
The Growth conditions are those described in Table 1. Autotrophic medium with nutrient (M
a1
) were
prepared with synthetic wastewater, composed of the following (per liter): NaCl, 7mg; CaCl
2
, 4 mg;
MgSO
4
·7H
2
O, 2 mg; K
2
HPO
4
, 21.7 mg; KH
2
PO
4
, 8.5 mg; Na
2
HPO
4
, 33.4 mg and NH
4
Cl, 3 mg [16].
Nitrogen-limited media did not contain NH
4
Cl.
The mixotrophic growth of both cases, (M
c
and M
c1
) had the initial composition of M
a
and M
a1
respectively. In this case 10 g L
-1
of dextrose monohydrate have been added to M
c
and M
c1
[11, 17].
pág. 4538
Table 1 Growth mediums for Chlorella sp.
Media
Description of the medium
M
a
Nitrogen-limited synthetic wastewater.
M
a1
Synthetic wastewater with nitrogen.
M
c
Nitrogen-limited synthetic wastewater with glucose.
M
c1
Synthetic wastewater with nitrogen and glucose.
Growth was carried out for 21 days in glass bottles. Two replicates were established for each medium,
all with a total volume of 100 mL, of which 10 mL corresponds to the portion of the inoculum
mentioned above and a cell density of 1.2 x 10
6
~
1.9 x 10
6
cell mL
-1
. With a light intensity variable
from 1000 to 5000 lux at a 12:12 photoperiod of the light / dark cycle without the addition of external
CO
2
.
Cell count
Cell growth monitoring was carried out every 48 hours until reaching the stationary phase, collecting
1 mL aliquots for each treatment. Counting was performed using a Neubauer camera and an optical
microscope with a 40 x objective. The cell concentration of each treatment was calculated with the
equation [15]:





where
, is the average number of cells that exist in 1 mm
2
.
Quantification of Biomass
The recovery of the biomass was carried out by centrifugation for a period of 15 min at 4000 rpm. To
know the growth in terms of dry weight, a volume of 10 mL (in duplicate) was filtered on GF / a
filters, previously tared. Each filter was placed in the oven at a temperature of 105 °C for 4 hours,
they were subsequently weighed until the constant weight was determined [10]. The total dry weight
was obtained by the weight difference between the filters (empty and with sample), from the
following equation [18]:
󰇛

󰇜

where,
represent weight of filter with sample (mg),
weight of empty filter (mg) and
is
volume of filtered culture (L).
pág. 4539
Extraction and Quantification of Lipids
The lipid extraction was carried out by the modified Bligh and Dyer method. Using a mixture of
chloroform: methanol (1: 2) [15]. The quantification of the percentage was carried out using the
following equation [19]:




Removal of Nutrients
To know the consumption of nutrients, present in the growth medium, the methodology proposed by
Marin et al. [20] to measure the content of nitrogen present in the form of ammonium, and phosphate
in the form of orthophosphates, both at the beginning and at the end of the crop.
Statistical Analysis
The statistical analysis was carried out using Minitab version 19.2.0. The obtained data were analyzed
statistically to determine the degree of significance at probability (P) < 0.05 using analysis of variance
ANOVA.
Results and Discussion
The cell growth rate of Chlorella sp. is shown in Figure 1 for growth media M
a
(Figure. 1.a), M
a1
(Figure. 1.b), M
c
, (Figure. 1.c) and M
c1
(Figure. 1.d). It is possible to see that the growth rate was
significantly higher for both media. When the species did not present nitrogen limitation (M
a1
and
M
c1
), the cell concentration reached values of 5.30 x 10
6
± 1.36 x 10
5
and 3.14 x 10
7
± 1.95 x 10
6
cell
m L
-1
, respectively. Likewise, it is observed that the highest cell concentrations are found in all the
mediums with a light flux of 3000 lux, having exponential growth from the eighth day.
pág. 4540
Figure 1 Chlorella sp. growth kinetics in the media M
a
(a), M
a1
(b), M
c
(c) and M
c1
(d), subjected to
light fluxes of 1000, 3000 and 5000 lux
The significant differences evaluated in this work about the specific growth rates, and the final cell
density obtained in both media are agree with the results obtained by Rosales et al. [21]and Ortiz et al.
[22]. Also, the main disadvantage of the physiological stress strategy by nutrients is associated with
the reduced cell division and, therefore, the low generation of cells per day. These values can be seen
in Table 2.
Table 2 Maximum kinetic growth parameters of the Chlorella sp., in each of the treatments
Growth
medium
Final cells
concentration
(cells mL
-1
)
Specific growth
rate
(generation day
-1
)
Generation
time (day)
Divisions
per day
M
a
2.84 x 10
6
± 1.85 x 10
5 *
0.08
a
41.1
0.1
M
a1
5.30 x 10
6
± 1.36 x 10
5
0.12
a
10.4
0.1
M
c
2.23 x 10
7
± 1.13 x 10
6
0.24
b
33.8
0.3
M
c1
3.14 x 10
7
± 1.95 x 10
6
0.28
b
22.1
0.4
* Average of two repetitions. ** The means (± standard error) within each column without common superscript differ
significantly at P <0.05, performing an analysis of variance (ANOVA).
pág. 4541
Figure 1 (d) shows that on the fourth day M
c 1
, an exponential growth begins , while the graphs refer
to the medium M
c
, which presents an average of 1.80 x 10
7
± 2.12 x 10
6
cells m L
- 1
, in the same
phase.
Until now, the study variables did not show significant changes in the treatments. By the middle of the
experiment, the growth of the species in M
c1
had increased by approximately 25%, with a cell average
of 2.43 x 10
7
± 9.89 x 10
5
cells m L
-1
, compared to an increase in 18.5% of the medium M
c
, with an
average of 1.84 x10
7
± 1.05 x 10
6
cells m L
-1
. The significant difference points to what was previously
indicated by other authors [11], even working in a mixotrophic medium, the stimulus that affects cell
growth to a greater extent is nitrogen deficiency in the medium, since, the lack of physiological
conditions prevented the increase in cell division, as does the present work, reaching day 7 of 16 in
experimentation.
In the present work, the M
c
and M
c1
media (mixotrophic conditions), obtained higher concentrations
when compared with M
a
and M
a1
(autotrophic conditions).
This can be attributed to the fact that glucose used as a source of organic carbon in this work, was
easily metabolized by microalgae, a situation that concurred with that presented by Rodríguez et
al[11]
Biomass Concentrations
Chlorella biomass yield has been reported in the ranges between 400 ~ 800 mg L
-1
, as reported by
Castillo et al. [23]. Figure 2 shows the results of the biomass concentration in media with nitrogen (M
a
and M
c
) and with limited nitrogen (M
a1
y M
c1
). The mixotrophic medium with nitrogen (M
c1
) gave the
highest yield, with an average of ~ 530 mg L
-1
as indicated in Table 3. Some authors report that
Chlorella presents better yields in medium with enrichment in organic carbon and the presence of
nitrogen. (mixotrophic), compared to autotrophic medium, reporting values between 500 and 150 mg
L
-1
, respectively [11].
pág. 4542
Figure 2 Biomass concentrations of Chlorella sp. in each of the treatments exposed to different light
intensities.
Liang et al. [7] attribute the previous behavior to the willingness of species like Chlorella to modify
their metabolic pathway with the presence of sugars. Additionally, Lang et al. [17] mention that the
use of glucose for the cultivation of microalgae in mixotrophic conditions promotes an increase in
biomass production, as presented in this research.
Table 3 Maximum biomass yield (mg L
-1
) of Chlorella sp. species in the most favorable growth
medium (3000 lux).
Growth medium
Peak performance
*
(mg L
-1
)
M
a
135.71 ± 10.10
** a
M
a1
164.29 ± 10.10
a
M
c
478.56 ± 10. 10
b
M
c1
535.71± 20.20
c
* Average of two repetitions. ** The means (± standard error) within each column without common superscript differ
significantly at P <0.05, performing an analysis of variance (ANOVA).
The averages of the maximum yield of the autotrophic medium M
a
and M
a1
did not present
significant differences. However, there is a notable disparity between the autotrophic and mixotrophic
media, as is the case of Ma and Mc, with a difference of ~350 ± 200 mgL
-1
and in the M
a1
and
M
c1
media with a difference of ~370 ± 272.74 mg L
-1
which represents more than 100%. Li et al [24],
report in their research that the biomass concentration doubled when there was nitrogen in the culture
medium and a source of organic carbon, a scenario that is shared in the present work. This
phenomenon is also explained by Freitas et al. When they mention that the formation of mixotrophic
pág. 4543
cultures with the help of organic carbon compounds accelerates the metabolism of species such as
Chlorella sp., as well as their cellular composition [25].
In the M
c1
medium, the yields exceeded 500 mg L
-1
, using only half the glucose than other reported
works [11] . The 50% saving in the use of the organic carbon source, with respect to the reference,
speaks of a cost reduction in the production of microalgae from this research.
It can be affirmed that the samples with higher light intensity favored a greater production of CO
2
and,
with it, the obtaining of better cell concentrations and biomass [25]. As can be seen in Figure 2, the
difference between cultures exposed to 1000 and 3000 lux ranges from 45 ~ 50 % in the case of
autotrophic cultures and from 2 ~ 6 % for mixotrophic cultures. Some authors mention that the
limitation of light prevents an efficient photosynthetic conversion and favors the appearance of shade
gradients, a problem that affects cell density such a situation did not occur in this investigation [25,
26].
Overexposures then favored substantial increases in culture temperature, as well as excessive stress,
which led to photooxidation and photoinhibition in the case of all experiments exposed to 5000 lux,
thus proving that a variation in culture temperature for species like Chlorella, ranges from 20 ~ 30%
[27, 28]
The difference in biomass increase between culture techniques can be attributed to what was
described by Izadpanah et al., mentioning the biological compatibility of the species such as Chlorella
sp. Between the isolation medium and the growth medium is noticeable. Since, mimicking
environments similar to the one used in the isolation of the species favors and efficient the growth of
the microalgae. Since, in this work, the species was found in an autotrophic medium of isolation [26].
Lipid Concentration
According to the analysis carried out, Figure 3 shows that the M
a
and M
a1
(autotrophic) medium has
lower lipid yields than the M
c
and M
c1
(mixotrophic) medium, with percentages ranging between
20~30 % and 25~46%, respectively.
pág. 4544
Figure 3 Lipid concentrations of Chlorella sp. in each of the treatments exposed to different light
intensities
Also, Liang et al. [7] report that in an autotrophic medium with nitrogen limitation, Chlorella
presented a yield close to 30 % against 36% in a mixotrophic medium using 1% v/v of organic carbon
source. Such results, in the first instance, are similar to those of the present investigation since Ma
achieved a lipid yield greater than 30% and secondly, the yield of M
c
is 10% higher than that of that
reference.
Table 4 Maximum lipid yield (mg L
-1
) of the Chlorella sp., in the different growth media
Growth medium
Peak performance
(mg L
-1
)
M
a
*
40.36 ± 6.96
**a
M
a1
23.36 ± 7.12
a
M
c
244.60 ± 4.41
b
M
c1
204.40 ± 9.11
b
* Average of two repetitions. ** The means (± standard error) within each column without common
superscript differ significantly at P <0.05, performing an analysis of variance (ANOVA).
The analysis of the information in Table 4, showed that the mediums M
a
and M
a1
(autotrophs) do not
present significant differences between them, but there is a disparity within the mixotrophic
mediums. We can attribute this to the composition of the mediums since M
c
and M
c1
are mediums with
the addition of organic carbon in the form of simple sugar, while the remaining mediums are purely
autotrophic. Rodríguez et al. [11], report that Chlorella yields in mediums with compositional
pág. 4545
differences, such as those reported in the present investigation, range between 6 ~ 12% for purely
autotrophic medium, and 16 ~ 38% for mixotrophic medium. In both cases, the mediums reported
here surpass such results. It is important to point out that M
a
and M
c
show better results in lipid
accumulation compared to M
a1
and M
c;
Such effect was sought after from the beginning of the
research, since according to Castillo et al. [23] subjecting species such as Chlorella to nutrient stress
would directly impact fatty acid synthesis, thus increasing lipid production and achieving a maximum
yield.
The analysis of the information in Table 4, showed that the M
a
and M
a1
(autotrophic) media do not
present significant differences between them, but there is a disparity within the mixotrophic media.
This can be attributed to the composition of the media, since M
c
and M
c1
are media with added
organic carbon in the form of simple sugar, while the rest of the media are purely autotrophic. Some
authors have reported that Chlorella yields in media with compositional differences, such as those
reported in the present investigation, oscillate between 6 ~ 12% for a purely autotrophic medium and
16 ~ 38% for a mixotrophic medium [11]. It is important to note that M
a
and M
c
show better results in
lipid accumulation results than to M
a1
and M
c
. This effect was sought from the beginning of the
investigation, since according to Castillo et al. [23], subjecting species such as Chlorella to nutritional
stress would directly impact fatty acid synthesis, thus increasing lipid production and achieving
maximum yield.
Although the limitation of nitrogen, exclusively to decrease cell division (Ma and Mc), this restriction
made it possible to redirect the synthesis of CO2 inside the cell, converting it mainly into neutral
lipids. In 2020, Feng et al. found that crops with sufficient N stored a lower amount of lipids than
those limited in this nutrient, a situation shared by both media in this research [29]. High light
intensities are known to modify the growth rate and the composition of the resulting biomass. Some
authors suggest that crops with an increase in simple sugars and medium intensities of white light
(1500-2500 lux) potentiate the content of carbohydrates and lipids [25]. Such a case is presented for
the Mc and Mc1 cultures, wich present an increase of 24 and 20% in lipid content, respectively, when
going from 1000 to 3000 lux of light intensity, and only a difference of between 8 and 10%. For
exposures from 3000 to 5000 lux, in the same cases.
pág. 4546
It can be observed that after the cultures exposed to 3000 lux, those that 5000 present better lipid
yields and this can be attributed to the fact that the overexposure of light allowed better of
synthesizing the macro and micronutrients of each culture, in the initial stage of the experiment ,
declining for exactly the same reason once the species was acclimatized to each growth technique [28]
[29].
Removal of Ammonium and Phosphate
According to the analysis, the mediums M
a
and M
a1
did not present significant differences in the initial
content of both parameters; a similar case occurred in the mediums M
c
and M
c1
. After the culture time
had elapsed, the removal values for each of the mediums were varied, see Table 5 The residual
mediums did not show significant differences among themselves. For the cases of M
a
and M
c
, the
initial and final values of nitrogen concentration in ammonium form were similar. The results are
associated with the fact that both media were designed with nitrogen limitation, and it is presumed
that the data reported here are due to the fixation of such compounds in the synthetic growth medium
by the species [30]
Table 5 Initial characterization of the culture media for the growth of Chlorella sp. Ammonium
(
) and phosphate (

)
Growth medium

(PPM)


(PPM)

(PPM)


(PPM)
Initial
Initial
Final
Final
M
a
0.99 ± 0.04
a
*
1.50 ± 0.04
**
0.85 ± 0.03
*
1.38 ± 0.01
**
M
a1
1.07 ± 0.03
a
1.64 ± 0.13
0.97 ± 0.01
0.98 ± 0.01
M
c
4
1.75 ± 0.04
b
1.42 ± 0.08
0.87 ± 0.04
1.34 ± 0.02
M
c1
5
1.16 ± 0.01
b
1.42 ± 0.02
0.87 ± 0.01
0.99 ± 0.04
* Average of two repetitions. ** The means (± standard error) within each column without common superscript differ
significantly at P <0.05, performing an analysis of variance (ANOVA).
Ramos et al. [31], reports in their work removal of ammonium

, of 21.48%, with a synthetic residual medium, while in the present investigation similar removal
values were reached in the media M
c
and M
c1
with 25.1 and 24.3 % respectively. In the case of the
removal of total dissolved phosphate, the highest values were presented for M
a
and M
c
, with 40 and
30% respectively. García et al. [32], mention in their research that the good assimilation of this
nutrient is directly related to the metabolic processes of microalgae that cause the biomass growth of
the species.
pág. 4547
CONCLUSIONS
Chlorella sp. was able to demonstrate its adaptability by growing in both autotrophic and mixotrophic
media, the latter being the most viable option in terms of total energy yield, as it was able to store a
percentage of lipids within the range already reported, while the amount of organic matter managed to
harvest is not compromised. However, for future work it is important to mention that the choice of the
culture medium will depend on the objectives that the work itself is planned. It should be noted that
the biomass-lipid ratio required being symbiotic for this work due to its energetic implications.
For the specific case of this work, it is considered that a medium lacking in some nutrients, for
example, nitrogen, presented better results in terms of cell concentrations, biomass and lipid yield,
compared to an enriched medium, where both macro and micronutrients are available. In this sense, a
parallel economic benefit is offered, since it is said that microalgae can be cultivated with a lower
economic requirement than that already reported.
Finally, the potential of the Chlorella sp. species was not limited to energy fines; In addition, its great
capacity to be used as an ecological agent in the treatment and bioremediation of wastewater will be
confirmed. This opens the way to a biorefinery concept, an innovative sector with a great impact in
the area of biotechnology recently. This methodology would be producing biomass with future uses in
biofuels (biodiesel, bioethanol, biohydrogen), bioremediation of wastewater and simultaneously,
products with high added value (biopolymers, biofertilizers, pigments, etc.).
BIBLIOGRAPHIC REFERENCES
[1] M. A. Mohammad Mirzaie, M. Kalbasi, S. M. Mousavi y B. Ghobadian, «Investigation of
mixotrophic, heterotrophic, and autotrophic growth of Chlorella vulgaris under agricultural
waste medium,» Preparative Biochemistry & Biotechnology , vol. 46, nº 2, pp. 150-156,
2016. DOI: 10.1080/10826068.2014.995812
[2] A. M. Ardila Alvarez, Y. Lopez Matos, M. Vasquez Caceres, A. Gonzalez Delgado y A.
Barajas Solano, «Obtaining lipids and carbohydrates from microalgae via design of selective
culture media,» Tecno Lógicas, vol. 20, nº 38, pp. 83-94, 2017. ISSN 0123-7799
[3] G. Salas Herrera, A. Benavides Mendoza, A. Zermeño Gonzalez , A. Orta Davila y F. D. J.
Sanchez Perez, «Evaluation of microalgae for the production of economically useful biomass
pág. 4548
using produced water.,» Revista Mexicana de Ciencias Agrícolas, vol. 12, nº 12, pp. 2423-
2435, 2015. ISSN 2007-0934
[4] G. Acien, A. Reis, G. Torzillo y E. Molina Grima, Photobioreactors for the production of
microalgae, Almería, España: Latest Edition, 2017. ISBN 9780081010235,
https://doi.org/10.1016/B978-0-08-101023-5.00001-7.
[5] S. Banerjee y S. Ramaswany, «Dynamic process model and economic analysis of microalgae
cultivation in open raceway ponds.,» Algal Research, vol. 26, pp. 330-340, 2017. ISSN 2211-
9264, https://doi.org/10.1016/j.algal.2017.08.011.
[6] P. Rodriguez Ramos, Y. Sanchez Miranda, L. Zumalacarregui de Cardenas, O. Perez Ones,
A. Hernadez Milan, P. Echeveste de Miguel y A. T. Lombardi, «Biomass production from
Chlorella vulgaris microalgae in bubble column photo-bioreactors,» Afinidad IQS, vol. 73, nº
574, 2016. ISSN 0001-9704
[7] Y. Liang, N. Sarkany y Y. Cui, «Biomass and lipid productivities of Chlorella vulgaris under
autotrophic, heterotrophic and mixotrophic growth conditions,» Biotechnol , vol. 31, pp.
1043-1049, 2009. https://doi.org/10.1007/s10529-009-9975-7
[8] L. L. Estévez Landazábal, A. F. Barajas Solano, C. Barajas Ferreira y V. Kafarov,
«Improvement of lipid productivity on Chlorella Vulgaris using waste glycerol and sodium
acetate.,» Ciencia, Tecnología y Futuro, vol. 5, nº 2, pp. 113-126, 2013.
https://doi.org/10.29047/01225383.203
[9] R. A. E. Fatah Hamouda, N. Mohamed Sorour y D. S. Yeheia, «Biodegradation of crude oil
by Anabaena oryzae, Chlorella kessleri and its consortium under mixotrophic conditions,»
International Biodeterioration & Biodegradation, vol. 112, pp. 128-134, 2016.
https://doi.org/10.1016/j.ibiod.2016.05.001
[10] A. M. Silva Benavides, «Evaluation of agricultural fertilizers on the productivity of
microalgae Chlorella sorokiniana.,» Agronomía mesoamericana, vol. 27, nº 2, pp. 265-275,
2016. https://doi.org/10.15517/AM.V27I2.24361.
[11] G. Rodríguez Castillo, C. Amarelo Santos, M. Guerrero Barrantes y A. Delgado dos Reis,
«Study of morphological and phisiological characteristics of Chlorella protothecoides
pág. 4549
oriented to lipid production for biofuel.,» Tecnología en marcha, vol. 29, nº 3, pp. 3-11, 2016.
http://dx.doi.org/10.18845/tm.v29i6.2897.
[12] T. S. Lin y J. Y. Wu, «Effect of carbon sources on growth and lipid accumulation of newly
isolated microalgae cultured under mixotrophic condition.,» Bioresource Technology, vol.
184, pp. 100-107, 2015. https://doi.org/10.1016/j.biortech.2014.11.005
[13] H. . A. Hasan, M. . H. Muhamad, B. Ji, N. . A. Nazairi, . K. . W. Jiat, S. . I. S. W. A. Sim y A.
. F. M. S. Poh, «Revolutionizing wastewater treatment with microalgae: Unveiling resource
recovery, mechanisms, challenges, and future possibilities.,» Ecological Engineering, vol.
197, p. 107117, 2023. https://doi.org/10.1016/j.ecoleng.2023.107117
[14] M. Ortiz Moreno, C. Cortés Castillo, J. Sánchez Villarraga, J. Padilla y A. Otero Paternina,
«Evaluating microalgae Chlorella sorokiniana growth in different culture mediums in
autotrophic and mixotrophic conditions,» ORINOQUIA, vol. 16, nº 1, pp. 11-20, 2012. ISSN
0121-3709
[15] B. O. Arredondo Vega y D. Voltolina, Métodos y Herramientas Analíticas en la Evaluación
de la Biomasa Microalgal., Baja California Sur: CIB, 2015. ISBN 978-607-7634-21-8
[16] X. Liu, K. Wang, J. Zhang, J. Wang, J. Wu y F. Peng, «Ammonium removal potential and its
conversion pathways by free and immobilized Scenedesmus obliquus from wastewater,»
Bioresource Technology, vol. 283, pp. 184-190, 2019.
https://doi.org/10.1016/j.biortech.2019.03.038
[17] X. Lang, Z. Liu, M. Xu, L. Xie y R. Li, «Effects of glucose on photosynthesis and growth of
Chlorella sp. HN08 cells,» Wei Sheng Wu Xue Bao, vol. 57, nº 4, pp. 550-559, 2017.
ISSN/ISBN: 0001-6209
[18] P. Santhakumaran, S. . K. Kookal, L. Mathew y J. G. Ray, «Bioprospecting of Three Rapid-
Growing Freshwater Green Algae, Promising Biomass for Biodiesel Production,» BioEnergy
Research, vol. 12, pp. 680-693, 2019. https://doi.org/10.1007/s12155-019-09990-9
[19] M. Amin y P. Chetpattananondh, «Enhanced Lipid Recovery from Marine Chlorella Sp.,»
BioEnergy Research, vol. 12, pp. 665-679, 2019. https://doi.org/10.1007/s12155-019-09986-5
pág. 4550
[20] A. Ruíz Marín, L. Mendoza Espinosa y T. Stephenson, «Growth and nutrient removal in free
and immobilized green algae in batch and semi-continuous cultures treating real
wastewater.,» Bioresource Technology, vol. 101, nº 1, pp. 58-64, 2010.
https://doi.org/10.1007/s12155-019-09986-5
[21] A. Rosales Quintero y R. Meza Gordillo, «Efecto del fotoperiodo y privación de nutrientes en
la producción de lípidos, clorofila y proteína en la microalga Chlorella vulgaris,» Chetumal,
Quintana Roo, México., 2018.
http://repositorio.digital.tuxtla.tecnm.mx/xmlui/handle/123456789/3057
[22] M. Ortiz Moreno, C. Cortés Castillo, J. Sánchez Villarraga y J. Padilla, «Evaluating
microalgae Chlorella sorokiniana growth in different culture mediums in autotrophic and
mixotrophic conditions,» ORINOQUIA, vol. 14, pp. 230-240, 2012. ISSN 0121-3709.
[23] O. Castillo, S. Torres Badajoz, C. Núñez Colín, V. Peña Caballero, C. Herrera Méndez y J.
Rodríguez Núñez, «Biodiesel production from microalgae: progress and biotechnological
prospects.,» Hidrobiológica, vol. 27, nº 3, pp. 337-352, 2017. ISSN 0188-8897.
[24] X. Li y H. X. Xu, «Large-scale biodiesel production from microalga Chlorella protothecoides
through heterotrophic cultivation in bioreactors,» Biotechnol Bioeng, vol. 98, pp. 764-771,
2007. DOI: 10.1002/bit.21489
[25] B. C. Freitas Bastos de , A. P. Cassuriaga Aguiar, M. Morais Greque De y J. A. Costa,
«Pentoses and light intensity increase the growth and carbohydrate production and alter the
protein profile of Chlorella minutissima,» Bioresource Technology, vol. 238, pp. 248-253,
2017. https://doi.org/10.1016/j.biortech.2017.04.031
[26] M. Izadpanah, R. Gheshlaghi, M. Akhavan y A. Elkamel, «Effect of light spectrum on
isolation of microalgae from urban wastewater,» Algal Research, vol. 29, pp. 154-158, 2018.
https://doi.org/10.1016/j.algal.2017.11.029
[27] A. Baran Sozmen, A. Atá y B. Övez, «Optimization of the algal species Chlorella miniata
growth: Mathematical modelling and evaluation of temperature and light intensity effects,»
Biocatalysis and Agricultural Biotechnology, vol. 2022, p. 102239, 2022.
https://doi.org/10.1016/j.bcab.2021.102239
pág. 4551
[28] A. Baidya, T. Akter, M. Rabiul Islam, A. Azad Shah, M. Amzad Hossain, M. Abdus Salam y
S. Indra Paul, «Effect of different wavelengths of LED light on the growth, chlorophyll, β-
carotene content and proximate composition of Chlorella ellipsoidea,» Heliyon, vol. 7, p.
e08525, 2021. https://doi.org/10.1016/j.heliyon.2021.e08525
[29] P. Feng, Z. Xu, L. Qin, M. Asraful Alam, Z. Wang y S. Zhu, «Effects of different nitrogen
sources and light paths of flat plate photobioreactors on the growth and lipid accumulation of
Chlorella sp. GN1 outdoors,» Bioresource Technology, vol. 301, p. 122762, 2020.
https://doi.org/10.1016/j.biortech.2020.122762
[30] A. Gulghe, F. A. Ansari, P. Singh y F. Bux, «Heterotrophic cultivation of microalgae using
aquaculture wastewater: A biorefinery concept for biomass production and nutrient
remediation.,» Ecological Engineering, vol. 99, pp. 47-53, 2017.
https://doi.org/10.1016/j.ecoleng.2016.11.013
[31] R. Ramos y R. Pizarro, «Growth and bioremediation capacity of Chlorella vulgaris
(Trebouxiophycea, Chlorophyta) cultivated in wastewater generated in the fish farming of the
yellowtail amberjack Seriola lalandi,» Biología Marina y Oceanografía, vol. 53, nº 1, pp. 75-
86, 2018. http://dx.doi.org/10.4067/S0718-19572018000100075.
[32] C. C. García Gozalbes, Z. Arbib y J. A. P. Vargas Machuca, «Growth Kinetics and Nutrient
Uptake of Microalgae in Urban Wastewaters with Different Treatment Levels.,» Water
Technology and Sciences, vol. 6, nº 1, pp. 49-68, 2015. http://hdl.handle.net/10498/19683