Journal of CO2 Utilization 64 (2022) 102153

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Journal of CO2 Utilization

journal homepage: www.elsevier.com/locate/jcou

Biofixation of CO2 and biomass production from model natural gas using
microalgae: An attractive concept for natural gas sweetening
Zahra Khoobkar a, Hossein Delavari Amrei b, *, 1, Amir Heydarinasab a,
Mohammad Ali Mohammad Mirzaie c
a
Department of Chemical Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran
b
Department of Chemical Engineering, Faculty of Engineering, University of Bojnord, Bojnord, Iran
c
Department of Chemical and Polymer Engineering, Yazd University, Yazd, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: Despite the fact that natural gases are the most vital source of energy, pollutants like carbon dioxide in natural
CO2 fixation gases cause sediment and erosion of equipment, catalysts poisoning, and the most important environmental
Natural gas pollution. Microalgae also have the ability to remove CO2 from some gas streams. In this work, for the first time,
Gas sweetening
a species of microalgae is used to fix carbon dioxide from the natural gas stream that contain 7% (v/v) CO2.
Microalgae
Therefore, the effect of different strategies of model gas injection, light intensity, and light/dark cycle on the
Chlorella sp
biomass productivity, carbon dioxide fixation rate, and lipid and chlorophyll content of Chlorella sp. were
investigated. The results showed that when the model gas was injected in the dark period, biomass productivity
and carbon dioxide fixation rate reached the highest level. In point of fact, under light intensity of 300 µmol
photon m-2s-1 the fixation rate has reached 240 mg L-1 day-1. In addition, both using nitrogen as a model gas
balancer and higher level of light intensity arose increased productivity and fixation. Also, the combination of
increased light intensity and model gas injection in the dark cycle provided a condition in which higher rates of
lipid accumulation were induced. The highest lipid content observed in this condition was 42% obtained.
Aeration using nitrogen as a model gas balancer decreased lipid production.

1. Introduction
Global demand for energy, fossil fuels, natural gas, and liquid fuels is
rapidly increasing, due to population growth. Pollutants like carbon
dioxide in natural gas and its derivatives are responsible for larger is­
sues. Sediment and erosion of equipment, catalysts poisoning and
deactivation in chemical process and environmental pollution can be
mentioned as side effects. Therefore, removing pollutants from natural
gas like sour gas streams is vital [1].
As a result, removing impurities such as carbon dioxide from natural
gas is crucial. The acceptable level of carbon dioxide in natural gas is 2%
Molar and 50 ppm in liquid gas (LNG) [2,2]. Nowadays, various ways
are used to remove carbon dioxide from natural gas. Adsorption-based
technologies in which solid adsorbents are used for CO2 removal from
a gas mixture. [3]. Due to the high density of carbon dioxide in sour gas,
the process is not recommended on a large scale and current absorbents
have limited selectivity and absorption capacity. Therefore, they are not
able to remove CO2 entirely [4]. Acid gas removal from sour gas by
cryogenic is another tool. In this phenomenon, sour gas which contains a
large amount of carbon dioxide is purified in a distillation unit without
freezing. This method is cheaper in comparison with other chemical and
physical removal methods [5], while, higher energy consumption and
extravagant instruments are considered disadvantages [3,6]. Mem­
branes are another viable candidate for CO2 removal from sour gas. But
CO2 doesn’t absorb entirely due to the low selectivity of membranes and
it requires a sweating process [7]. Moreover, Alkanolamine solutions are
used by industries to remove acid gas from synthetic, natural, and
refined gases. The merit of this method is the ability to the reduction of
acid gas density at low pressures. In general, since the absorption pro­
cess with chemical solvents is processed at low pressure, they consume
lower energy in comparison with physical solvents in which higher
pressures are required [8]. Massive environmental pollution and unex­
pected transformation in chemical solvents are demerits of this method.
Additionally, they are overpriced and extra operation in the process is

* Corresponding author.
E-mail address: h.delavari@ub.ac.ir (H. Delavari Amrei).
1
P.O. Box 94531–1339

https://doi.org/10.1016/j.jcou.2022.102153
Received 6 May 2022; Received in revised form 13 July 2022; Accepted 20 July 2022
Available online 29 July 2022
2212-9820/© 2022 Elsevier Ltd. All rights reserved.
Z. Khoobkar et al. Journal of CO2 Utilization 64 (2022) 102153

needed to recover them. Table 1

Compare to chemical fixation, biological CO2 fixation by microalgae Different strategies based on the type of gas injection and the light/dark
is a promising and novel tool to reduce CO2 emission [9–11]. Microalgae cycle.
generate organic materials by converting sunlight into active chemicals Strategy Condition
and CO2 into sustainable chemicals without emitting pollutants [12]. a Model gas injection for 24 h
Advantages such as higher photosynthetic capacity contrary to plants 24 light / 0 dark
(15–20 fold) [13], shorter growth cycle (4–10 days), higher flexibility b Model gas injection in the lighting period
[14], the ability to use seawater and wastewater for cultivation [15], N2 injection in the dark period
12 light / 12 dark
and inevitable competition with farm products lead to global attention
c Model gas injection in the dark period
for microalgae. 50% of microalgae biomass is carbon and 1.83 Kg CO2 N2 injection in the lighting period
can be fixed by only 1 Kg microalgae [16,17]. In addition, microalgae 12 light / 12 dark
generate biofuels or high additive products through the uptake of CO2
[18].
Using microalgae to reduce CO2 emission from stacks is dealing with
various problems and according to studies the high amount of CO2 leads X=490×OD560 (1)
to the insufficient growth rate of microalgae. Because microalgae enter − 1
The specific growth rate (μ, day ) was calculated using Eq. (2):
in a lag phase when a high density of CO2 is introduced and the growth
rate decreases the lower rate is due to lower pH caused by the higher ln XX0t
mass of CO2 [19,20]. Even those species that resist a higher amount of μmax = (2)
t
CO2 showed a prolonged growth cycle which is a sign of low biocatalyst
activities of enzymes in cells that disrupt CO2 conversion. Where Xt is the concentration of biomass (mg L− 1) at time t and X0 is the
Stripped natural gas from the Khangiran gas field in Iran contains 7% concentration at the beginning of cultivation. The biomass productivity
carbon dioxide which is an acceptable amount to cultivate microalgae (P, mg L− 1 day− 1) was obtained using the following equation,
[21]. Moreover, microalgae are photosynthetic organisms in which light Xf − X0
plays an essential role in growth parameters [22,23]. Therefore, in this P= (3)
tf − t0
work, the removal of carbon dioxide from a model natural gas stream,
containing 7% (v/v) CO2, in a photobioreactor containing microalga Where Xf is the biomass concentration on the last day of the experiment
Chlorella sp. was investigated. Previous studies have mostly focused on and tf demonstrates time [26].
removing carbon dioxide from flue gases and biogas, and removing acid According to the molecular formula of microalgae biomass, CO0.48
gases from natural gas flow has not received attention and investigation. H1.83 N0.11 P0.01 [27], CO2 fixation rate (FCO2, mg L-1 day-1) obtaining
Furthermore, the effect of different strategies of the model gas injection, from following equation [28]:
light intensity, and light/dark cycle on the algal growth and CO2 bio­
fixation have been considered. In fact, the main purpose of this work is CO2 fixation rate (FCO2) =1⋅88×P (4)
to provide the basic concept of natural gas sweetening by microalgae
and production of high value biochemicals from algal biomass.

2.3. Model natural gas

2. Materials and methods
The model natural gas of this study is made according to the analysis
2.1. Microorganism
of the extracted natural gas from the Khangiran gas field located in Iran.
This gas is included 88% methane and 7% CO2. Air or nitrogen is added
The microalga Chlorella sp. (Chlorella sp.; PTCC6010, Persian Type
to this gas by 5%. The rate of injection of the model gas has been
Culture Collection) was prepared from the Iranian Research Organiza­
regulated by a mass flow meter with a flow rate of l L h-1. (LAMBDA CZ s.
tion for Science and Technology (IROST) and used to test the CO2 fix­
r.o. 61400 Brno. Czech).
ation efficiency. This microorganism was cultivated in Rudic’s medium
(pH~8) [24]. The medium contained (in mg L− 1) 300 NaNO3, 20
2.4. CO2 analysis
KH2PO4, 80 K2HPO4, 20 NaCl, 47 CaCl2, 10 MgSO4 7H2O, 0.1 ZnSO4
7H2O, 1.5 MnSO4 H2O, 0.08 CuSO4 5H2O, 0.3H3BO3, 0.3
Individual hydrocarbon detection in the samples generally could be
(NH4).6Mo7O24.4H2O, 17 FeCl3.6H2O, 0.2 Co(NO3)2H2O, and 7.5 EDTA
detected using a gas chromatograph equipped with a flame ionization
[24].
detector (GC/FID). In this research, it was used to determine the CO2
The highest consumption rate of microalgae is 200–600 mg L-1day-1
concentration at the inlet and outlet of the reactor (GC-2010 Plus-
while some species of Chlorella sp. can assimilate 800–1000 mg L-1day-1.
SHIMADZU). Due to the presence of moisture in the outlet gas, after
They remove sulfur dioxide, nitrogen oxide, and organic compounds, as
passing a silica gel chamber, the outlet gas flow of the photobioreactor
well [25]. Borkenstein et al. used stack gas of a cement factory to culture
enters the basket to be dehydrated. In the next step, the collected gas in
Chlorella emersonii under the photoautotrophic condition in 2010. They
the baskets is injected into the gas chromatograph.
reached 3.3 g L-1 CO2 consumption and 2.06 g L-1 biomass production in
prolonged cultivation. Doucha et al. have reported investigating stack
gas emitted by burnt natural gas to culture Chlorella sp. using a thin 2.5. Light Source
layered photobioreactor in an open space.
A white LED lamp (NAMA LIGHT, Iran) was used as a light source.
This lamp covers all wavelengths of light in the range of 400–700 nm.
2.2. Growth parameters
2.6. Cultivation in a Bubble Column Photobioreactor
Optical density (OD) was measured at a wavelength of 560 nm by a
UV/Vis spectrophotometer (Philler Scientific, SU6100). The relationship After the pre-cultivation stage in an Erlenmeyer flask of 500 ml
between the concentration of biomass (X, mg L-1) or the dry cell mass (working volume 250 ml) and under white fluorescent light with an
and OD560 was obtained according to the following equation [26]: intensity of 100 μmol photons m− 2 s− 1, the microalga Chlorella sp. was

2
Z. Khoobkar et al.
Fig. 1. Schematic diagram of the process and operational condition.
Table 2
Different strategies based on gas composition and light intensity.
2 − 1
Strategy Light intensity (μmol photons m− s ) Gas composition
1 100 CH4: 88%, CO2: 7%, Air: 5%
2 100 CH4: 88%, CO2: 7%, N2: 5%
3 300 CH4: 88%, CO2: 7%, Air: 5%
4 300 CH4: 88%, CO2: 7%, N2: 5%
5 100 CH4: 100%
6 300 CH4: 100%
7 100 CO2: 100%
8 300 CO2: 100%
transferred to a bubble column photobioreactor (diameter:15 cm,
length: 70 cm) containing 7500 ml of Rudic’s culture medium with a
capacity of 10 L (8 L working volume). To inject the model gas into the
reactor, three strategies are considered. Also, light/dark cycles have
been different in each strategy. In Table 1, these strategies (a, b, and c)
are presented (Fig. 1).
Also, according to the composition of the injected gas and light in­
tensity, 8 strategies were considered, which are presented in Table 2. In
these strategies, in addition to the effect of model gas, the effect of pure
CH4 and pure CO2 has also been investigated.
Based on the presented strategies in Tables 1 and 2 and considering
all cases, 24 experiments were performed, which were named as follows:
S1a, S2a, S3a, S4a, S5a, S6a, S7a, S8a; S1b, S2b, S3b, S4b, S5b, S6b,
S7b, S8b; S1c, S2c, S3c, S4c, S5c, S6c, S7c, S8c.
2.7. Measurement of Chlorophyll and lipid Content
To measure the absorption spectrum of extracted pigments, 2 ml of
the sample was centrifuged, the liquid layer above the deposited
microalga was removed, and instead, 2 ml of methanol was added to the
sample container. The sample was placed in an ultrasonic bath for
45 min. Then, pigments together with 2 ml of methanol were placed in
the ice bath overnight and then centrifuged for 10 min at 2500 ×g to
separate the cell residues. Ultimately, to determine the content of
chlorophyll a, the absorbance of the sample was measured at 662 and
645 nm wavelengths using UV/Vis spectrophotometer and the following
equation [26]:
Chlorophyll a (Ca ) = 11.75 • OD662 − 2.350 • OD645
Chlorophyll b (Cb ) = 18.61 • OD645 − 3.960 • OD662
By using Bligh and Dyer [29], total lipid content was calculated and
solvent in this experiment was chloroform-methanol.
Journal of CO2 Utilization 64 (2022) 102153
2.8. The effect of pure methane
The majority of natural gas is consists of methane, also considering
that methane may affect cell growth, therefore, Chlorella sp. has been
aerated by pure methane (cases: S5a, S6a; S5b, S6b; S5c, S6c).
2.9. The effect of pure CO2
The high level of CO2 usually affects the growth of microalgae
negatively. Exposure to the high concentration of CO2 makes microalgae
enter the lag phase which prevents growth. The reduction of growth is
likely due to a decrease in pH caused by a high concentration of CO2. The
first step of using microalgae to CO2 assimilation is selecting adaptable
species, tolerating high concentrations of carbon dioxide, performing
high enzymes activity, being able to cultivate easily in mass scales, and
concluding valuable material for postharvest applications [30]. Con­
cerning this point, Chlorella sp. was aerated by pure CO2 (cases: S7a, S8a;
S7b, S8b; S7c, S8c).
2.10. The effect of photorespiration
The effect of O2 in input gas has barely been studied, although the
high level of O2 to prevent saturation can be organized by sparging gas
or installing a gas exchanger in a photobioreactor. Model gas mixed with
air and pure N2 to evaluate the effect of O2 on photorespiration and
carbon fixation. The flow rate of this gas was obtained by a mass flow
meter to reach the fixed gas concentration.
2.11. Statistical Analyses
All experiments in this study were carried out in triplicate, in two
different runs. One-way analysis of variance (ANOVA) was performed
using (SPSS, V25) package for Social Science Software (Table S1). P
values < 0.05 were considered significant in differences.
3. Results and discussion
3.1. The effect of light regime
Light and CO2 mass transfer efficiency are key elements to address
for successful CO2 fixation systems. The light regime is the primary
(5)
factor in the efficiency of the photosynthesis process [31]. In general,
cell growth is directly dependent on light intensity and cycle. Unless the
(6)
high level of light damage the photosystem and result in photoinhibition
[32]. Since CO2 fixation by microalgae is performed under photoauto­
trophic condition, CO2 fixation of special species are related to cell
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Z. Khoobkar et al.
Fig. 2. Time course of cell growth for different samples in strategies S1 and S2.
Fig. 3. Time course of cell growth for different samples in strategies S3 and S4.
growth rate and efficiency of light consumption [31,33]. On the other
hand, the Calvin cycle reaction is a light-independent reaction in
photosynthesis. These reactions don’t directly assemble energy from
photons. The required energy of the Calvin cycle reaction is provided
with ATP and NADPH created by the energy of photons in
light-dependent reactions [8]. The light reactions take place in the
thylakoid membranes where the usable energy for the photosystem is
stored and transferred to molecules, Adenosine triphosphate (ATP), and
Nicotinamide adenine dinucleotide phosphate (AADPH).
̅̅→
2H2 O + 2NADP+ + 3ADP + 3Plight 2NADPH + 2H+ + 3ATP + O2
NADP , ADP, and P are an abbreviation of oxidized NADPH, Aden­
+ conditions convert carbon into sugar and starch through a pentose
osine diphosphate, and phosphor, respectively. To convert carbon di­
oxide to carbohydrates, ATP and NADPH are used in a darkness
experiment [34].
3CO2 + 9ATP + 6NADPH + 6H+
→ C3 H6 O3 + P + 9ADP + 8P + 6NADP+ 3H2 O
It is shown that in the process of carbon assimilation, light doesn’t
play an important role. Therefore, in cases S1c, S2c, S3c, S4c, S5c, S6c,
S7c, and S8c the efficiency of carbon fixation has been assessed in the
dark phase. In addition, the effect of light with intensities of 100 μmol
photons m− 2s− 1 and 300 μmol photons m− 2s− 1 on growth, CO2 fixation
rate, and cellular chlorophyll content has been investigated. The growth
Journal of CO2 Utilization 64 (2022) 102153
Fig. 4. Time course of cell growth for different samples in strategies S5 and S6.
Fig. 5. Time course of cell growth for different samples in strategies S7 and S8.
curves are shown in Figs. 2, 3, 4, and 5. The results show that in case S4c
the highest level for growth, specific growth rate, and biomass produc­
tivity was achieved. These values were 1798.3 mg L-1, 1.00, and 127.61,
respectively. Besides, the highest level of CO2 fixation equal to
239.90 g L-1d-1 is obtained from this case (Table 3). It should be
considered that in addition to changes in biomass production and CO2
fixation, biomass created in different light conditions has different
biochemical compounds. Microalgal cells cultured in low light under
photoautotrophic conditions absorb carbon to synthesize amino acids
(7) and other cellular parts. However, cells growing in saturated light
phosphate-reducing pathway [31]. Some researches prove that
compared to continuous lighting conditions, light cycle 12:12 (D/L) is
more effective in microalgal growth [35]. Because light is discontinuous
(day and night cycle) and some species of microalgae affected by their
natural habitat pick special light condition and their photosynthesis and
(8)
growth rate do not depend on continuous lighting [35]. On the other
hand, Toro [36] has investigated the growth of microalgae Chaetoceros
gracilic and Isochrysis galbana in two light cycles of 0:24 and 12:12 (D/L).
He doubled the light intensity of 12:12 and showed the cell growth rate
of the two systems is equal. Consequently, it could be concluded that
cellular growth is not only affected by the light cycle but the level of
light energy in the cycle, as well.
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Table 3 differs from one species to another. Each microalgal species select a
Algal growth parameters and CO2 fixation rate for different cases. unique mechanism that permits environmental compatibility [41].
Cases P (mg L-1 day-1) µmax (day-1) FCO2 (mg L-1 day-1)
3.3. Lipid content
S1a 52.36 ± 0.11 0.34 ± 0.00 98.43 ± 0.01
S1b 56.21 ± 0.02 0.37 ± 0.00 105.67 ± 0.02
S1c 59.36 ± 0.00 0.40 ± 0.00 111.59 ± 0.00 The results show that the use of light with more intensity has
S2a 75.35 ± 0.02 0.57 ± 0.00 141.66 ± 0.00 significantly increased the efficiency of cellular lipid production (Fig. 7).
S2b 77.56 ± 0.00 0.50 ± 0.00 145.81 ± 0.57 So in the case S3c, compared to the similar system that received less light
S2c 108.36 ± 0.02 0.71 ± 0.00 203.71 ± 0.00
S3a 59.01 ± 0.00 0.31 ± 0.00 110.93 ± 0.01
intensity, cell lipid accumulation shows a 31% increase. Also, a light/
S3b 64.96 ± 0.01 0.41 ± 0.00 122.12 ± 0.00 dark cycle (12 h/12 h) was more efficient than continuous exposure,
S3c 67.06 ± 0.01 0.57 ± 0.00 126.07 ± 0.01 and lipid accumulation in a gas-aerated cases in the dark cycle was
S4a 83.16 ± 0.01 0.65 ± 0.00 156.34 ± 0.02 higher than in all cases.
S4b 97.16 ± 0.92 0.68 ± 0.00 182.66 ± 0.00
The presence of light is an important trigger for lipid synthesis in
S4c 127.61 ± 0.01 1.00 ± 0.00 239.90 ± 0.00
S5a 18.83 ± 0.01 0.29 ± 0.00 35.40 ± 0.01 microalgae. The amount of light absorbed by surfaces depends on a variety
S5b 25.44 ± 0.00 0.34 ± 0.00 47.83 ± 0.00 of factors, including the light source, the intensity, the spectra, the dark-
S5c 30.24 ± 0.00 0.35 ± 0.00 56.85 ± 0.00 light photoperiods, frequency, and the amount of light exposed to the
S6a 19.77 ± 0.00 0.30 ± 0.00 37.17 ± 0.02 surface [44–47]. They have a particular impact on the microalgae biomass
S6b 25.65 ± 0.00 0.28 ± 0.00 48.23 ± 0.01
S6c 33.70 ± 0.00 0.36 ± 0.00 63.36 ± 0.00
formation rate and the biochemical content, including lipids, carbohy­
S7a 6.16 ± 0.11 0.34 ± 0.00 11.58 ± 0.01 drates, proteins, vitamins, pigments, and antioxidants [48]. The lipid
S7b 2.66 ± 0.03 0.36 ± 0.00 5.00 ± 0.00 amount of Chlorella sp. and Monoraphidium sp. grew when the light in­
S7c 23.31 ± 0.00 0.28 ± 0.00 43.82 ± 0.00 tensity has been risen from 40 to 400 μmol photons m− 2 s− 1 while suitable
S8a 0.56 ± 0.00 0.07 ± 0.00 1.05 ± 0.00
fatty acids profiles have been obtained to produce biodiesel [44]. Also, Wu
S8b 0.38 ± 0.00 0.06 ± 0.00 0.72 ± 0.00
S8c 26.95 ± 0.00 0.30 ± 0.00 50.67 ± 0.01 et al. [48] obtained a similar result in Gracilaria lemaneiformis when they
increased the light intensity from 20 μmolm− 2 s− 1 to 200 μmolm− 2 s− 1.
Generally, high light intensity enhances neutral storage lipid synthesis
3.2. Chlorophyll content whereas low light intensity promotes membrane lipid synthesis [49].
The studies of Atta et al. [50] found that the lipid content of Chlorella
Information on cellular chlorophyll content is presented in Fig. 6. vulgaris decreased by 13% when the photoperiod was increased from a
Although pigments are responsible for receiving light, according to re­ 12–16 h light period at the same light intensity. Higher luminance
sults curves of chlorophyll content do not entirely coincide with growth causes the lipid content to decrease due to increased chloroplastidial
curves because antenna protein of chloroplast in the process of the light- activity to prevent photochemical damage.
receiving, store light energy and transfer it to the light system and play a It should be noted that the use of nitrogen gas as a model gas balancer
vital role in the light reaction [37]. Reducing light-harvesting complex harmed cell lipid accumulation, so all cases have shown a downward
(LHC) units and level of microalgae chlorophyll cells may result in a trend in lipid accumulation throughout the culture period. The use of
decrease in the yield of receiving light and reducing wasted excitation pure methane gas for aeration did not significantly affect cellular lipid
energy and consequently raise production of biomass. [38–40]. accumulation, and the lipid production process was almost constant.
Increasing light intensity from 100 to 300 μmol photons m− 2s− 1 Lipid accumulation decreased dramatically when pure carbon dioxide
contributed to elevating chlorophyll content production. It has been was used for aeration.
proved that cases S1a, S1b, S1c, S3a, S3b, S3c (Figs. 2 and 3) showed an Due to the conversion of unused CO2 to carbonic acid (H2CO3), high
increase in chlorophyll in the first week but a reduction in the second CO2 levels lower the pH of the culture medium. A decreased pH can
week of cultivation. While the highest chlorophyll content production inhibit microalgal growth, despite a greater carbon supply, since
belongs to a system that has been aerated non-continuous (case S3c). RuBisCO activity is inhibited in the process of concentrating CO2 [51].
Supposedly, continuous lighting surpassed discontinued lighting in
chlorophyll production when air is used to balance model gas. More­ 3.4. The effect of photorespiration
over, according to Figs. 2 and 3 results showed that when N2 is selected
as a model gas balancer (cases S2a, S2b, S2c, S4a, S4b, S4c), cellular Growth rate elevated without oxygen and, using nitrogen as a model
chlorophyll content go down. In general, the increase in biomass comes gas balancer in cases S2c and S4c increased by 81% and 89% growth
with a decrease in chlorophyll production. There are various reports on compared to cases S1c and S3C in which air was a model gas balancer.
reduction in chlorophyll and other carotenoids in systems with a high Although elevating light intensity from 100 to 300 μmol photons m− 2s− 1
level of CO2 concentration. For example, research showed when pure air increased biomass production, it seems the effect of removing oxygen
was aerated, the chlorophyll content reached a higher level than the from gas flow had a higher impact on growth efficiency. In addition,
condition that 6% CO2 was injected [28]. It can be concluded that a removing oxygen affects productivity (P) and maximum specific growth
reduction in pH arising from the high concentration of CO2 lowered rate (µmax) positively and all systems showed higher P and µmax in lack of
cellular chlorophyll content. While oxidative stress caused by lower pH oxygen. Notably, injection of gas without oxygen in the dark cycle had a
produces active oxygen species such as H2O2 which negatively affect better impact on growth parameters case S2c and P and µmax were 82%
cellular chlorophyll production [41]. Very acidic solutions and high and 72% greater than case S1c. These amounts in S4c were 89% and
concentrations of CO2 will decrease the rate of photosynthesis and the 78% greater than case S3c. Also, results indicate the efficiency of CO2
reason is the deactivation of key enzymes of the Calvin cycle caused by fixation affected by removing oxygen increased namely S2c compared to
acidification of the chloroplast stroma (stromal compartment chloro­ case S1c had 82% and S4c in comparison with S3c had 90% increase in
plast) [42]. Additionally, some researchers have concluded elevated CO2 CO2 fixation rate.
affects photosynthesis [43] and Promotes cyclic electron transport The presence of oxygen in entry gas could be a potential limitation of
around the photosystem to facilitate ATP production. Notably, CO2 cell growth [52]. With the assumption that carboxyl and Rubisco en­
impact is closely related to species, as well. Therefore, strong pieces of zymes uptake CO2 from the Calvin cycle to convert carbon into bio­
evidence suggest a decrease in pH caused by an increase in the avail­ energy, the excess of oxygen could be a real issue in microalgae
ability of CO2 will reduce cellular pigments like chlorophyll. However, cultivation because not only does limit the speed of photosynthesis but
CO2 compatibility with a high level of CO2 is a complex process that oxygen free radicals could also be toxic and damage cell membrane [53].

5
Z. Khoobkar et al.
Fig. 6. Time course of chlorophyll content (g−g−chlorophyll
biomass × 100 vs.
Several strategies have been suggested to remove excess oxygen that
remained in the system including the increase in the collision, stripping
culture medium with air, or neutral gas (such as Argon). Pulz et al.
(2001) reported that the balance of CO2 and O2 is an essential element to
reach a higher photosynthesis rate [53]. While the lack of O2 solution in
the environment facilitates carbon absorption (for example when ni­
trogen is used instead of air). On findings indicate that when N2 is used
to balance model gas, cellular chlorophyll content decrease. Generally,
an increase in biomass comes with a decrease in chlorophyll and much
research reported the reduction of chlorophyll and carotenoids in the
exposure of higher CO2 levels. For example, in a study when pure air was
aerated the chlorophyll content had a higher increase in comparison
with the time 6% of CO2 was used [28]. Lowering of pH level arising
from a higher level of CO2 reduces cellular chlorophyll. Under this
circumstance, due to reaching sufficient carbon, the cell growth peaked
at the highest level. While oxidative stress caused by lower pH generated
active oxygen species such as H2O2 that negatively affected the cellular
chlorophyll [41].
Journal of CO2 Utilization 64 (2022) 102153
day) for different strategies; A: S1 and S2, B: S3 and S4, C: S5 and S6, D: S7 and S8.
According to the current analysis harvested gas from a gas field
usually lacks or contains an insufficient level of oxygen. Therefore,
natural gas could be considered as a rich source of carbon.
3.5. Reduce the percentage of CO2 from the model natural gas
CO2 removal and fixation are two separate concepts. CO2 removal is
a process in which the main goal is removing carbon dioxide completely
to improve the quality of gas. While at the CO2 fixation the microor­
ganisms consume the carbon dioxide and convert it to valuable inner
cellular components. It could increase the cellular growth efficiency of
the microorganism and promote the quality of the stripped gas. There­
fore, the quality promotion of the gas is a major purpose in the CO2
fixation process. In the current study, perusing CO2 fixation from the
model natural gas. To assure that CO2 is fully consumed by microalgae,
used a GC-FID device for gas analysis. Reports indicate that microalga
Chlorella sp. can uptake carbon dioxide in the flow of the gas model
within 24 h and no CO2 has been detected in the outlet gas flow of
6
Z. Khoobkar et al.
Fig. 7. Time course of lipid content for different strategies; A: S1 and S2, B: S3 and S4, C: S5 and S6, D: S7 and S8.
photobioreactors. This analysis has been hired in S1c, S2c, S3c, S4c
cases.
3.6. Economic potentials
In order to use natural gas, excess CO2 must be removed from the gas
flow. So far, various methods have been used to remove CO2 from nat­
ural gas, including absorption, adsorption, membrane process, and
cryogenics [54,55]. It is worth noting that all the mentioned methods
collect CO2 in a very concentrated form, then the collected CO2 must be
discharged. In many cases, this concentrated CO2 is injected deep into
the earth or oceans, which has many environmental consequences[56,
57]. Among these consequences, the decrease in the pH of the oceans
can be mentioned, which destroys many species living in the oceans
[58]. Although the economic potential of the project is of great impor­
tance in the process of biofixation of CO2 using microalgae, in general,
first of all, the purpose of the CO2 biofixation process is to reduce the
harmful effects of chemical processes. Due to the fact that the process
details of this work have not yet been completed, a full and compre­
hensive discussion about its economic potential cannot be given. How­
ever, in the process of CO2 biofixation from natural gas using
7
Journal of CO2 Utilization 64 (2022) 102153
microalgae, high-value biomass is produced that can have some appli­
cations in various industries. However, the chemical process of natural
gas sweetening consumes high energy, seriously damages the environ­
ment, and has no other achievements.
The capacity and efficiency of microalgae-based processes for CO2
fixation and its conversion into valuable products depend on the
selected culture system, microalgae strain, and photobioreactor capac­
ity. The open cultivation system has a lower initial cost (€50/m3), but
the risk of contamination in such a system is high. Additionally, the
efficiency of using CO2 in such a system is at most 30%. Closed photo­
bioreactor systems have a higher construction cost (2000 €/m3), but the
risk of pollution and evaporation in these systems is reduced, and the
efficiency of using CO2 will be more than 95% [59]. The most important
parameters related to the stage of microalgae production include the
land area, biomass productivity, working days per year, and reactors’
expenses. In CO2 biofixation, the type and cost of water consumption
should be considered. Some types of microalgae have the potential to
grow in salty water, so if the gas facility is located near the sea, then
seawater can be used for microalgae cultivation. Also, in the economic
estimation, the cost of nutrients should be taken into account. The dif­
ference between the CO2 biofixation and the chemical removal of CO2 is
Z. Khoobkar et al.
Table 4
Price of different grade of algal biomass [59].
Algal Biomass Price Cultivation system
Low value biomass for production of Jet ~1000 €/t Open raceway
fuel, Bio-ethanol, Bio-fertilizer, …
Medium value biomass for production of ~10,000 Open raceway &
bioplastic, biopesticides, animal feed, … €/t Closed PBR
High value biomass for production of ~100,000 Closed PBR
β-carotene, Astaxanthin, … €/t
the production of biomass with high added value in the former. The
produced biomass will increase the economic potential of the project
due to its wide range of applications. Table 4 shows the price of biomass
produced from microalgae [59]. In general, biofixation of natural gas
using microalgae not only removes CO2 in an environmentally friendly
process, but carbon dioxide absorbers, the same microalgae that have
valuable biomass, increase during the removal process.
4. Conclusion
The current study has investigated the efficiency of removing carbon
dioxide from stripped natural gas using microalga Chlorella sp. to pro­
duce valuable cellular compounds such as pigments and lipids. The re­
sults indicated that removing oxygen from entry gas increased cell
growth and efficiency of CO2 fixation but decreased cellular pigment
and lipid content. Increasing light intensity and model gas injection in
the dark cycle enhanced lipid accumulation. Since natural gas lacks or
contains an insignificant level of oxygen, it could be considered an ideal
feed for microalga Chlorella sp. This study is the first-ever investigation
assessing the various gas injection regimes in a dark cycle which proves
carbon fixation is facilitated in the dark phase. A new idea of biological
sweetening of natural gas has been introduced in this article that can
remove CO2 from natural gas biomass while using it as biofuels. Future
studies can evaluate biomass fatty acids profiles to produce microalgae-
based biofuels after sweetening natural gas.
CRediT authorship contribution statement
Zahra Khoobkar: Investigation, Resources, Writing – original draft,
Formal analysis. Hossein Delavari Amrei: Conceptualization, Meth­
odology, Supervision, Writing – review & editing. Amir Heydarinasab:
Supervision, Writing – review & editing. Mohammad Ali Mohammad
Mirzaie: Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data Availability
The data has been used is not confidental.
Acknowledgements
Several thanks go to our English editor, Miss Marzieh Karimi
Mousivand.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.jcou.2022.102153.
8
Journal of CO2 Utilization 64 (2022) 102153
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