Algal Research 66 (2022) 102810

Contents lists available at ScienceDirect

Algal Research
journal homepage: www.elsevier.com/locate/algal

Genetic improvement of microalgae for enhanced carbon dioxide

sequestration and enriched biomass productivity: Review on CO2
bio-fixation pathways modifications
P. Priyadharsini a, N. Nirmala a, S.S. Dawn a, A. Baskaran b, P. SundarRajan c, K.P. Gopinath d,
J. Arun a, *
a
Centre for Waste Management, International Research Centre, Sathyabama Institute of Science and Technology, Jeppiaar Nagar (OMR), Chennai 600119, Tamil Nadu,
India
b
Department of Biotechnology, B. S. Abdur Rahman Institute of Science and Technology, GST Road, Vandalur, Chennai 600 048, Tamil Nadu, INDIA
c
Department of Chemical Engineering, Saveetha School of Engineering, Chennai, Tamil Nadu 602105, India
d
Department of Sustainable Engineering, Saveetha School of Engineering, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, Tamil
Nadu 602105, India

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

Keywords: Algae, carbon dioxide and wastewater are the most promising research topics in recent years. Integrated sus­
Microalgae tainable approaches on cultivating microalgae in both real-time and simulated wastewater with carbon dioxide
Carbon dioxide sequestration (CO2) are experimented as environmentally feasible approach. However, in terms of high macromolecules rich
Bio-fixation
biomass productivity (example: high lipid accumulated biomasses productivity for biofuel applications) are
Genetic engineering
Wastewater
needed to achieve sustainable productivity. Hence, it is needed to improve photosynthesis process by which we
Algae biomass can achieve higher CO2 sequestration and enriched biomass productivity. Therefore, on improving photosyn­
thesis process, efficiency of enzymes involved in photosynthetic pathways needs to be enriched, light utilization
range needs to be improved and majorly the release of captured CO2 needs to be minimized. These modifications
are achievable through genetic engineering approaches in environmentally friendly way. Hence, this review
provides the detailed potential strategies that helps in enhancing the microalgae-based CO2 fixation, technical
insights into existing limitations and challenges, future research directions to achieve sustainable development.

1. Introduction to adopt preventative actions to reduce CO2 emissions [5].

Carbon dioxide (CO2) emissions have been steadily increasing in
recent years, and this cycle will expect to continue. More than 80 % of
global energy output is derived from the combustion of fossil fuels,
which emit massive amounts of CO2, making it one of the primary ele­
ments of greenhouse gas emissions (carbon dioxide, methane, nitrous
oxide, chlorofluorocarbons) accounting for 68 % of global emissions
[1,2]. The CO2 emitted as a result of fossil fuel burning is expected to led
to global climate change, rising cost of fuel and the insecurity of fuel [3].
Furthermore, the high level of CO2 emissions has had substantial effects
on physical, biological, and socioeconomic systems, such as rising
temperatures, resource depletion, agricultural productivity reduction
and rising sea levels [4]. As a result, it is critical to investigate more
feasible energy options in order to reduce dependency on fossil fuels and
Several CO2 capture techniques have been reported to date,
including metal-organic frameworks, carbon nanotubes, activated car­
bon and zeolites. Fig. 1 explains the available different techniques on
carbon dioxide sequestration process. These materials are still not
commercially available and have not been evaluated for large-scale use
[6]. Due to their benefits and capacity to grow in wastewater, algae are a
readily available and suitable bio resource for biofuel production [7].
Algae biomass has the following advantages: it can be grown in any type
of water source; For growth, it absorbs nutrients from wastewater and
CO2 from the atmosphere for photosynthesis; and it uses less water than
terrestrial plants and can grow the whole year [8].
CO2 bio-mitigation using photoautotrophic microalgae, particularly
green microalgae, has recently gained the concern of the public. Green
microalgae are the most commonly occurring group of aquatic

* Corresponding author.
E-mail address: arunjayaseelan93@gmail.com (J. Arun).

https://doi.org/10.1016/j.algal.2022.102810
Received 28 April 2022; Received in revised form 12 July 2022; Accepted 1 August 2022
Available online 6 August 2022
2211-9264/© 2022 Elsevier B.V. All rights reserved.
P. Priyadharsini et al.
microalgae [9]. They grow quickly without contending for productive
land [10], although they are convenient to cultivate photo-
autotrophically to achieve high cell density both indoors and outdoors
[11]. Furthermore, numerous green microalgal species have a greater
tolerance for high CO2 levels and can effectively fix CO2 from a variety of
sources in an environmentally friendly manner [9].
Photosynthetic microorganisms, such as cyanobacteria, can adapt to
and grow in a diverse variety of CO2 concentrations. The acclimation
process is mediated by a series of changes at variety of cellular levels,
including changes in the expression of genes involved in the CO2
concentrating mechanism's operation (CCM). The presence of a CCM
was discovered in the green alga Chlamydomonas reinhardtii and the
cyanobacterium Anabaena variabilis for the first time [12].
In order to significantly increase photosynthetic CO2 fixation capa­
bility, a breakthrough in the control of the Calvin cycle is essential. A
sequence of enzymatic processes is carried out by 11 enzymes in the
Calvin cycle, which is a fundamental part of plant and algal metabolism.
Carboxylation (carbon fixation), reduction, and regeneration are the
three key phases of this cycle, which provides substrates for carbohy­
drate biosynthesis by utilizing ATP and NADPH [13]. The Calvin cycle is
initiated by ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco)
carboxylating the CO2 acceptor molecule ribulose 1,5-bisphosphate
(RuBP). Carbon assimilation is primarily determined by its catalytic
properties. This enzyme, which plays a critical role in the Calvin cycle,
has been the subject of several engineering efforts in recent years to
increase photosynthesis [9].
In recent year, Genetic engineering has received lot of attention
because new powerful genetic tools are widely available, and the
genome can now be edited more precisely than ever before to meet our
needs. The metabolic approach, also known as synthetic biology, has
received considerable attention, as designing a metabolic process in a
system where one does not exist opens up new possibilities for micro­
algae industrialization [14].
There have been several studies on the metabolic engineering of
photosynthetic microorganisms, with many focusing on fatty acid and
isoprenoid biosynthesis routes, respectively. In contrast, engineering
recombinant proteins in microalgae requires strategically introducing a
foreign gene encoding the desired protein into the algal genome,
allowing high protein activity and targeting to a specific subcellular
location. More microalgal strains may be easily identified and tools
suited to their genetic code as genome sequencing costs decrease. As a
result, research in this subject is expanding, and more broadly applicable
genetic toolkits are being designed to enable the commercial production
Fig. 1. Different type of techniques employed for carbon dioxide sequestration process.
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Algal Research 66 (2022) 102810
of numerous species of microalgae [15].
2. Microalgae
Microalgae range in size from a few micrometers to hundreds of
micrometers and are unicellular or have a simplistic multicellular
framework, which accounts for their rapid growth rate. Microalgae are
photosynthetic prokaryotic or eukaryotic algae that can survive in a
various environment. Microalgae are used in a variety of fields,
depending on the type of microalgae used and the culture conditions
that allow for growth and biomass production [16]. Used to produce
wide range of required products such as foods, pharmaceutical, cos­
meceutical industries, as well as agriculture as biofertilizers and bio­
fuels. A diverse range of algal-derived molecules can be aimed for cost-
competitive production due to their effective resource utilization, nat­
ural genetic diversity and faster growth [15]. They biosynthesize a
diverse range of metabolites with various bioactive properties, including
polysaccharides, fatty acids, essential amino acids, polypeptides, pig­
ments, vitamins, and minerals [17].
Microalgae appear to be more photosynthetically efficient than
terrestrial plants and are efficient CO2 fixers. When compared to
terrestrial feedstock crops, algae have a number of advantages. Although
their growth requirements are simplistic when compared to terrestrial
ecosystems, it requires nutrients, phosphate sources, nitrogen, trace
metals, water, sunlight and CO2. They use these resources very effi­
ciently, resulting in high productivity with relatively low water use [18].
Moreover, Microalgae seem to be the most viable production facility,
offering a long-term solution for carbon capture and recycling. They
have the advantage of being potential leaders in environmental cleaning
because of their CO2 fixation capability, which is multiple times greater
CO2 per unit area than agricultural crops [19]. Thus, CCS using micro­
algae is regarded as a potential alternative to many existing CO2
abatement strategies, owing to the ability to generate carbon credits
[17].
3. CO2 mitigation by microalgae
Microalgae being a photosynthetic organism which has the ability to
utilize the atmospheric carbon dioxide (CO2) present in the atmosphere
as a sole source of carbon and accumulate lipid, produce and sugars to
produce energy in the presence of water and sunlight. The utilization of
microalgae which also helps to reduce the amount of greenhouses gases
which are generated by the industries. This process helps the microalgae
P. Priyadharsini et al. Algal Research 66 (2022) 102810

to convert the observed CO2 into a bioenergy, bioactive products and can be utilized at the region of where the wastewater is generated.
food products. The microalgae contain about 50 % of carbon content in Mostly, this algae cultivation system was found to be more suitable and
their biomass. On an average of about 1.65–1.8 g of CO2 is needed for sustainable through coupling the algae cultivation system with waste­
the synthesis of 1 g dried microalgae biomass which shows the demand waters for biomass production as well as for energy production [1].
of CO2 for the microalgae growth [20]. The different strains of micro­ In the closed photobioreactor, it has a larger optical cross-sectional
algae have various biochemical properties resulting in CO2 tolerance area for it to receive both the natural and artificial lights for their
and fixing up of CO2 from the atmosphere. Few of the literature study growth. In the closed photobioreactor, the microalgae can be cultivated
has represented the characteristics of microalgae species resulting in on continuous basis to achieve the high growth rate as well as high CO2
bio-fixation of CO2 with higher growth rate, tolerance of other gases like fixation. Parameter which affects the growth of the algae can be kept in
SOx and NOx, higher consumption of CO2 ability to grow wastewater. control, can maintain a stable culture conditions, can proceed with high
Microalgae are capable to trap the carbon more efficiently of about density cultivation, improves high CO2 fixation efficiency, reduced
10–50 times for their growth while comparing to the terrestrial plants. water evaporation and light intensity can be maintained are its major
Various microalgae species differs in the level of tolerance with respect advantages factor involved comparing with open pond system [24].
to the CO2 concentration from the flue gas [21]. Therefore, accordingly Other types of photobioreactors which are available for the micro­
based upon the tolerance of CO2, the microalgae species are divided as algae cultivation includes cylindrical airlift photobioreactors, tubular
CO2 sensitive species with less than 2–5 % CO2, species that tolerate CO2 photobioreactor, stirred fermenters, flat panel photobioreactors,
of 5–20 % are referred to as CO2 tolerant species, species that tolerate floating and bag reactors. Table 2 provides the detailed characteristics
CO2 of 20–100 % are referred to as extreme CO2 tolerant species. advantages and remarks on carbon dioxide sequestration. The other
Microalgae species like Arthrospira sp., Chlorella sp., N. oculate, B. braunii novel microalgae system for microalgae growth is offshore membrane
and S. dimorphus are the species which could tolerate the high amount of enclosures for growing algae (OMEGA) system. In the OMEGA system,
CO2 concentration. The bio mitigation of microalgae species at different the microalgae cultivated in the floating photobioreactors helps to
CO2 tolerance with their removal efficiency are reported in Table 1. deploy in protection of marine bay environment along with the
Other algae species of Scenedesmus exhibits the tolerance of high con­ municipal wastewater as well as the CO2 rich flue gas. The maintenance
centration of CO2 comparing with Chlorella sp. [22]. Whereas a mutant of uniform temperature and low fertilizer cost in the nutrient rich
strain of Chlorella sp. reports its ability to exhibit the high tolerance of wastewater and CO2 rich flue gas with low energy input are the main
CO2 concentration of about 70 %. The high level of CO2 consumption by advantages measures can be achieved through the OMEGA system
the microalgae have not affects the structure and functioning of the cultivation.
microalgae. Such symbiotic microalgae alter them accordingly for the In the recent times, the CO2 mitigation from flue gas by growing
appropriate use in the advanced technology in order to mitigate the CO2 microalgae in the closed photobioreactor involves analyzing the char­
[23]. acteristics of physiological conditions of microalgae in the closed pho­
tobioreactor, growth kinetics studies of microalgae, pilot scale
3.1. Cultivation systems for CO2 sequestration cultivation of microalgae and helps in the byproduct's development.
Screening and optimizing of efficient microalgae species are maintained
Carbon sequestration using microalgae have been implemented all in the closed photobioreactor system and for the scaling up of micro­
over the world for different purposes. Various cultivation systems have algae cultivation is proceeded with the open photobioreactor system.
been developed and implemented by understanding the cultivation The CO2 sequestration combined with microalgae cultivation by
conditions. capturing the flue gas from the nearer industries can be achieved [25].
The cost of scaling of closed photobioreactor system rises drastically due
3.1.1. Photobioreactor to its structure reconstruction, light collection and its distribution
Photobioreactors are used for the large cultivation of phototrophic throughout the system, cooling and heating system. So that, the closed
microorganisms. Based upon the exposure to the atmosphere, they are photobioreactor found to be comparative with the open photobioreactor
classified as open system and closed system. In the case of open system, system.
the CO2 is introduced into the system either directly by injecting or by
mixing it with the ambient air [16]. So that, the introduced CO2 dis­
solves in the culture media. Whereas, with respect to the improved open
pond system, a sump closes to the paddle wheel around the depth of 1.5
m is adopted to achieve the solubility of CO2 in water while direct in­ Table 2
jection or also to enhance the efficiency of CO2 fixation. The open pond Notable characteristics of closed reactors on carbon dioxide sequestration upon
system is built with simple construction with low cost and user friendly algae cultivation [26,27].
in operations. The disadvantages faced with open pond systems includes Reactor Notable characteristics Configuration remarks
unstable culture conditions, prone to high contamination, light intensity model
decays the medium depth, high evaporation loss etc. In order to over­
Vertical Higher gas exchange, Greater light Bigger bubbles, higher shear
come the is advantages of these open pond systems, the advanced open tube and dark cycles, less land required, effects, Higher CO2
pond systems are made with stacked multi-layered hybrid bioreactor. high cost, higher photosynthetic sequestration rate
This multilayered bioreactor is considered to be a small footprint system efficiency
Tubular High volumetric biomass density, CO2 utilization affected by
photoinhibition, large land space imbalance in CO2/O2
Table 1 required concentration due to dark and
Bio mitigation of CO2 using microalgae species. light cycles
Stirred Higher gas exchange, Greater Shear sensitivity can be
Microalgae Experimented CO2 Removal efficiency References
agitation and aeration model, improved by modification in
species concentration of CO2
desired hydrodynamic system, aeration and agitation process
C. vulgaris 8% 0.14 % [20] Greater light and dark cycles, less for better CO2 utilization
N. gaditana 8% 0.17 % land required, high cost, higher
Scenedesmus sp. 10.6 % 66 % [22] photosynthetic efficiency
C. pyrenoidosa 10 % 0.002 % [21] Flat plate Lesser power required, Low Imperfect mixing causes dead
S. obliquus 10 % 0.002 % photosynthetic efficiency, aeration pockets in flat panels
Anabaena sp. 15 % 93.8 % [23] results in cells by shear damage

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P. Priyadharsini et al. Algal Research 66 (2022) 102810

3.2. CO2 sequestration and biomass production Table 3

Literature collection on microalgae cultivated conditions towards lipid pro­
The CO2 capture for the microalgae biomass production which utilize duction with simultaneous carbon dioxide sequestration.
the CO2 for its growth and converts into energy and value-added Microalgae Cultivation Lipid Remarks References
products through photosynthesis is highly appreciable. The utilization conditions yield (%)
of microalgae biomass shall pose an important part in economics of Chlorella Polycarbonate 33–38 – [30]
microalgae-based carbon sequestration [28]. Table 3 provides the minutissima bottles, Air flow
detailed collection of articles that are reported on microalgae cultivation rate-0.2 LPM,
Constant
with simultaneous CO2 sequestration and biomass production. For the
fluorescent light
commercialization of microalgae production, the lack of efficient Chaetoceros Erlenmeyer 39.8 Better CO2 [31]
microalgae harvesting techniques and the economics are the major calcitrans flasks in orbital sequestration
challenges faced for the implementation. Enormous amount for energy shaker incubator, potential
is required as well as the harvesting of tiny microalgae about 1–70 μm Temperature- during lipid
25 ◦ C, synthesis
and the negatively charged microalgae cells was found to be cost Continuous process
effective of about 20 %–30 %. Centrifugation, sedimentation, air flota­ illumination-100
tion, electrophoresis, flocculation are the major harvesting techniques μmol photons/
used in microalgae biomass production. The harvesting of microalgae is m2/s, 2 weeks,
Input air
continued for the conversion of biomass into biofuels like biodiesel, bio
contained 5 %
crude oil, ethanol, hydrogen production as well as other value-added carbon dioxide
byproducts chemicals which is used in the cosmetics, food, and phar­ Ellipsoidion sp. Erlenmeyer 27.4 Absence of
maceutical industries through downstream processing [29]. The har­ flasks in orbital dark cycle
vested microalgae cells can be diverted for the biogas production by shaker incubator, and lesser
Temperature- capability to
performing anaerobic digestion and also for the production of biocrude
25 ◦ C, consume
oil production by performing hydrothermal liquefaction. The micro­ Continuous CO2a
algae biomass can also be used for the production of omega-3 fatty acids, illumination-100
fertilizer, biological active substances etc. μmol photons/
m2/s, 2 weeks,
Input air
4. Genetic engineering of microalgae contained 5 %
carbon dioxide
Microalgal biotechnologies are developed by performing coupling of CCAP 849/6 Combination of 30.9 –
CO2 sequestration and commercial production of microalgal biomass to aerobic/
anaerobically
enhance genetic engineering techniques. The primary CO2 fixing
treated swine
enzyme Rubisco helps to catalyse the oxygenation or carboxylation of wastewater
ribulose-1,5-bisphosphate during the Calvin cycle which has more pro­ (Ammonium-
teins universally among the photosynthetic organisms. While comparing 418.8 mg/L,
Nitrate-11.3 mg/
with other microalgae and plants, the Cyanobacteria Rubisco holds a
L, Phosphate-5.4
low affinity to CO2 and tend to develop Carbon dioxide concentrating mg/L) with 50 %
mechanism (CCM). This CCM plays a vital role in fixing carbon enzymes dilution,
available in carboxysomes like Rubisco and carbonic anhydrases. Thus, Temperature-
the available micro storage compartments help to enhance the CO2 level 25 ◦ C,
Continuous
surrounded by the Rubisco and tend to combine with other available
illumination-
substrate O2. In recent times, researchers have showed more interest in 45–50 μmol m− 2
targeting the selected gene specific to carbonic anhydrase and Rubisco s− 1, Air + CO2
metabolic pathway in fixing more carbon using genetically engineered supply (0.5 vvm
containing 2%v/
phototrophic organisms.
v)
The oleaginous microorganism isolated from the natural environ­ Chlamydomonas Municipal 25.5 Agitation and [32]
ment should holds a desired characteristics of possess robustness, fast reinhardtii wastewater, nutrient
growth rate, high lipid accumulation, capable to survive in any envi­ Biocoil availability
ronmental conditions and suitable for genetic manipulations. Mostly, photobioreactor, enhanced the
pH-7.5, Injection lipid content
the strains isolated from the natural environment are found not to be
of air and small
capable to meet all the desired characteristics. Therefore, the strains amount of CO2
improvement is required by performing classical or random mutagen­ Dunaliella Modified NORO 60.6–67.8 Great amount [33]
esis, genetic engineering and adaptive laboratory evolution (ALE). tertiolecta medium, pH-8, of lipid
Roux bottle, CO2 content in cell
Mutagenesis and ALE has greater advantages of inexpensive, requires
enriched air 0.25 grow in 3 %
less information of strains genetic composition, genetic regulation and LPM carrying 3 % CO2
biochemical pathway and also requires technical manipulation tools. CO2,
Temperature-
4.1. Random mutagenesis 30 ◦ C,
Illumination-150
μmol m− 2 s− 1
Random mutagenesis can be performed using physical, chemical Chlorella vulgaris Tubular 28–58 Nitrogen [34]
mutagens, insertional mutagenesis which leads to alterations of strains photobioreactor, limiting
DNA and selection of mutants with metabolic properties is done [37]. Liquid velocity- condition
0.63 m/s, Air helped higher
supply-5 LPM, lipid yield
4.1.1. Physical Mutagenesis Illumination-
The physical mutagens were performed by exposure of strains to (continued on next page)
radiation via ion beams, UV rays, X-and ƴ rays and atmospheric room

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P. Priyadharsini et al. Algal Research 66 (2022) 102810

Table 3 (continued ) Non alkylating reagents like formaldehyde, hydroxylamine, nitrous

Microalgae Cultivation Lipid Remarks References acid, methoxamine, bisulphite, hydrazine is used to cause crosslinking
conditions yield (%) between DNA and proteins and leads to deletions, deamination. The
130 μmol m− 2
alkylating agents like alkyl sulphates, N-nitroso compounds, methyl
s− 1, methane sulfonate (MMS), ethyl methane sulphonate (EMS), diethyl
Temperature- sulphate (DES) and N-methyl-N-nitro-N-nitrosoguanidine (NTG) were
25 ◦ C, 3 days, used for enhancing the lipid yield of the oleaginous microorganisms
Watnabe's
[15,38]. Alkylating agents add alkyl group to the hydrogen bond of
medium
(Nitrogen guanine and adenine base pairs and leads to N-glyosidic bond and un­
abundance)-Low dergoes hydrolysis to induce depurination and pairing errors in DNA.
nitrogen medium
UTEX 1185 Glass bubble 15.9–56 – [35] 4.1.3. Insertional mutagenesis
column
bioreactor,
Insertional mutagenesis involves in addition of exogenous DNA into
Continuous genomic DNA through non-homologous recombination using a plasmid
stirring, Bristol in an antibiotic resistance marker and leads to gene disruption, null
medium, mutation, alters the metabolic activity which leads to the accumulation
Temperature-
of higher amount of lipid in an oleaginous microalga.
30 ◦ C,
Continuous
illumination 150 5. CO2 bio-fixation pathway – enhancement strategies
μE m− 2 s− 1,
covering dark Different strategies have been attempted to enhance CO2 fixation of
cycle, 18 days
fermentation, Air
microalgae. Broad spectrum approaches like
supply – 0.5 vvm,
Enriched air (i) Increasing efficiency of enzymes that involved in the Calvin cycle
stream carrying pathways
5 % CO2
(ii) Manipulating light-harvesting photosynthetic apparatus by non­
Nannochloropsis Cylindrical glass 29.7–22.7 Higher lipid [36]
oculata photobioreactor, formation at photochemical quenching (NPQ)
Temperature- enhanced CO2 (iii) Incorporating one or more additional carbon fixing cycles to
26 ◦ C, input rate up reduce net carbon loss.
Illumination 300 to 15 % in air.
μmol m− 2 s− 1,
CO2 in input air
The Calvin cycle or Calvin-Benson-Bassham (CBB) cycle, a chain of
(0.2 LPM, 0.25 biochemical redox reactions, that converts CO2 and hydrogen carrier
vvm) into carbohydrate. Calvin cycle occurs in stroma of chloroplast, requires
a
CO2: Carbon dioxide, LPM: Liter per minute, vvm: volume of air sparged (in
9 mol ATP and 6 mol NAD(P)H for the fixation of 3 mol of CO2, found in
aerobic cultures) per unit volume of growth medium per minute. most eukaryotes and algae [39]. It involves in three stages: a carboxyl­
ation step executed by ribulose-1,5-bisphosphate carboxylase‑ox­
ygenase; a reduction of phosphoglycerate carried out by
temperature plasma. UV radiations between 250 and 290 nm induces
phosphoglycerate kinase (PGK) and NADP-dependent glyceraldehyde-3-
the formation deletion of thymine dimers which is responsible for the
phosphate dehydrogenase at the expense of ATP and NADPH; and a
transition of guanine and cytosine to adenine and thymine. The ion
regeneration phase that converts triose-phosphate into ribulose-1,5-
beams use heavy charged particles like 12C6+ which hold high linear
bisphosphate involving a series of enzymes (from triose phosphate
energy transfer helps in inducing higher mutation rates when compared
isomerase and fructose-1,6-bisphosphatase (FBPase) to phosphor­
to X and ƴ rays. Such heavy ion beams cause double strand breaks in
ibulokinase (PRK)) [40]. Figs. 2 and 3 explain the difference in genetic
DNA, small addition and deletion and base substitution. The low energy
engineering concept on carbon dioxide sequestration process in algae
nitrogen uses helps to damage the cell wall of the organisms and gen­
cells. The crux of the CBB cycle is the rate-limiting steps and this reaction
erates the free radicals within the cell and leads to DNA damage and
in mainly driven by the most putative photosynthetic enzyme “ribulose
mutation.
1,5 bisphosphate carboxylase/oxygenase (Rubisco)” which catalyzes
The ARTP uses ionized helium discharge plasma in a high frequency
reaction between CO2 and ribulose-1,5-bisphosphate (RuBP). The
electric field to induce mutation in the microorganisms. The radio fre­
turnover number (kc) of Rubisco, around 5 s− 1, and its catalytic effi­
quency is generated under 760 Torr atmospheric pressure and at 300 K
ciency (kc/Km) and affinity for CO2 are low, moreover it depends on the
temperature and applied on the surface of the microbial cells using
relative concentration of CO2 and O2 at the active site and specificity of
nozzle. ARTP causes damage to the DNA strands at a particular radio
the enzyme [41]. Due to increasing CO2 emission, fossil fuel combustion
frequency input, plasma distance, treatment time and helium rate. ARTP
causes severe impact on global warming, in order to prevent this, CO2-
method of mutagenesis techniques was found to be an advantage due to
concentration mechanisms have been adopted in several photosynthetic
it less operating cost and operational flexibility.
organisms, thereby enhancing CO2 concentration in Rubisco.
4.1.2. Chemical mutagenesis
In the chemical mutagenesis, they react with DNA and cause damage 5.1. Calvin cycle key enzymes advancement
in base pairing, transversions, deamination of purines and transitions,
frameshift mutations. The chemical mutagens are classified as Inter­ George Bowes discovered biologically important enzyme Rubisco,
calating agents, base analogs, non -alkylating agents and alkylating that initiates ribulose 1,5 bisphosphate reactions, depending on the
agents. The intercalating agents like acridine orange, proflavine, availability of the CO2 and O2, allowing the flow of flux between carbon
ethidium bromide and acriflavine is used to insert themselves in the releasing C2 pathway and carbon fixing C3 cycle [42]. Rubisco is a quite
DNA to cause a genetic frameshift mutation. The base analogs like 5- incompetent enzyme having very low affinity to atmospheric CO2,
bromouracil, 5- bromodeoxyuridine and 2- amino purine has similar having only 25 % of its catalytic activity, slow turnover rate (low Kccat).
chemical structure of nucleic acids which helps to cause base transitions. During oxygenation reaction, depending on O2 and CO2 concentration at
the active site of RuBP, a molecule of phosphoglycoate (2-PG) is

5
P. Priyadharsini et al.
Fig. 2. Sequestered carbon dioxide utilization sites in microalgae. Sequestered CO2 enters into the thylakoid and it is catalyzed to form 3-phosphoglycerate in
pyrenoids. Later it acts as processor to form pyruvate, fatty acids and starch. Pyruvate is the main component in TCA cycle. During photorespiration, oxygen is
catalyzed in Rubisco, phosphoglycolate is formed and CO2 is released.
produced together with one of phosphoglycerate (3-PGA) with reduc­
tion in the rate of net photosynthesis via photorespiratory pathway [43].
Most pioneering studies on CBB cycle and on RuBisCo have not only
focused on increasing the efficiency of the carboxylation reaction, but
also in association with synthesis and assembly.
Rubisco X-ray crystal structure revealed that rubisco from algae and
cyanobacteria is a hexadecameric complex having molecular mass of
560 kDa, with eight large subunit (rcbL; 50 kDa) and eight small subunits
(rbcS; 15 kDa). The catalytic site of a larger subunits arranged as a
tetramer of antiparallel dimers, capped by four small subunits at the top
and bottom [44]. A Japanese group successfully designed and generated
point mutation in SP8-T289 on rubisco enzyme from hyper thermophilic
archaeon Thermococcus kodakarensis, showed highest activity even at
high temperature both in vitro and in vivo conditions. Similarly, two
transplastomic tobacco variant were generated by inserting rubisco and
carboxysomal protein CcmM35 from the cyanobacterium Synechococcus
elongatus PCC7942 (Se7942), and found the rate of CO2 fixation per unit
of enzyme was higher than the wild type [45]. More interestingly,
hybrid rubisco approaches were developed by combining small subunits
from spinach, Arabidopsis and sunflower with algal large subunits by
electroporating in C. reinhardtii mutant that lacks rbcS gene. Significant
increase in CO2/O2 specificity was noted, but however, the Vmax value
retains normal. Hybrid mutant strains displayed reduction in photo­
synthetic growth and lacks chloroplast pyrenoids, indicating that rbcS
protein is essential for catalytic performance of rubisco [46]. Advance­
ment in carbon fixation efficiency by enhancing the catalytic activity of
rubisco has been implemented and established in most higher plants.
Additionally, fusion of integrative omics with genetic engineering tools
can refine the enzyme activity with higher performance [47]. On the
other hand, rubisco activity (Rca) was also monitored under steady-state
turn conditions and global curve fitting. The catalytic efficiency raised
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Algal Research 66 (2022) 102810
8.4-fold upon cooperative binding of a second magnesium ion that sta­
bilizes and mediates the formation of closed hexameric toroids capable
of high turnover rates. Alternatively, overexpression of RCA in micro­
alga Nannochloropsis oceanica exhibited significant elevation in rubisco
activity, increased photosynthesis and improved productivity of both
biomass and oil under low CO2 concentration [48]. In addition to this
several crucial enzymes that involved in CCB cycle is of greater impor­
tance, which includes sedoheptulose-1,7-bisphosphate (SBPase) and
fructose 1,6 bisphosphate aldolase (aldolase). SBPase is a functional
enzyme profound to exist in low level, involved in ribulose-1,5-
bisphosphate (RuBP) regeneration. Numerous targeted overexpression
studies on SBPase displayed, elevated photosynthetic efficiency and
Calvin cycle intermediates, higher CO2 fixation and biomass yield.
Correspondingly, overexpression of SBPase in microalgae Dunaliella
bardawil and Euglena gracilis were noted with increased glycerol pro­
duction, enhanced photosynthesis, biomass and wax ester productivity
[49–51]. Another essential enzyme is aldolase, where improved photo­
synthetic rate (2-fold) and over growth was reported in Chlorella vulgaris,
when expression levels of aldolase increased [9]. Although, diverse ge­
netic strategies have been implemented in tobacco plants, cyanobacteria
and microalgae, precise editing and expression of biologically signifi­
cant enzymes in the carbon fixation pathway and, the integration of
desired sequence of interest into particular microalgal genome remains a
strenuous task nowadays.
On the contrary, another group of enzymes called carbonic anhy­
drases (CAs) that involved in CO2 concentrating mechanism (CCM),
transports inorganic carbon to Calvin cycle [52]. When the CO2 gradient
fluctuated, CA amplifies to improve photosynthetic efficiency in rubisco.
CCM associated genes and its over-expression studies were reported
[53], the main CCM components includes CA (CAH1, CAH2, CAH3,
CAH4, CAH5 and CAH6), inorganic carbon transporters like (HLA3,
P. Priyadharsini et al.
Fig. 3. Schematic representation of engineering strategies towards reducing the carbon and energy loss. Towards improving the CO2 fixation, 6-phosphogluconate
dehydrogenase can be used as an alternate for Rubisco. In Calvin cycle, either it can be used in existing pathway or Crotonyl- CoA/ethylmalonyl-CoA/
Hydroxybutyrul-CoA can be used. Towards bypassing photorespiration process, instead of glycerate pathways which produce CO2, non Co2 producing pathways
like Arabinose-5P, Ribulose-1P, Xylulose and Erythrulose can be incorporated.
LCI1, LCIA, CCP1, CCP2 CemA, RHP1 and RHP2), CO2 uptake (LCIB,
LCIC, LCIA and LCIE), two nuclear transcriptional regulators (CIA5/
CCM1 and LCR1) and a SET domain methyl transferase (CIA6). Out of
which LCI1 and HLA3 mainly facilitates carbon uptake when CO2 is
limited. On the other hand, gene manipulation, mutation and regulation
studies on CCM were described [54]. Knocking out the specific HLA3
mRNA and CAH5 gene, resulted decrease in Ci uptake function. How­
ever, when dTALE, a synthetic transcriptional activator found to induce
HLA3 in Chlamydomonas, leading in increase biomass productivity,
growth rate and Ci uptake systems. Quite recently, molecular compo­
nents of CCM in 12 different alkaliphilic cyanobacteria were explored
using genome-based analysis, and the comparative analysis showed,
they share a common genes ccmKLMNO cluster, necessary for β-car­
boxysome assembly and function [55,56].
5.2. Manipulating NPQ mechanisms
Fine tuning or optimizing the light signaling pathways can be greatly
achieved by manipulating NPQ and increasing resistance to oxidative
stress. Generally photosynthetic organisms prevent oxidative stress from
light energy absorbed in excess through several photoprotective mech­
anisms. A major component is thermal dissipation of chlorophyll singlet
excited states and is called “nonphotochemical quenching (NPQ)”.
Modification on NPQ system can increase crop productivity, enhanced
carbon uptake, tolerance to rapid light intensities and biomass produc­
tivity, by altering excess sunlight via dissipating some light energy as
heat [57]. More interestingly, light-harvesting complex stress-related
(LHCSRs) proteins, identified as quencher during NPQ induction of
both photo systems (PSI and PSII). LHCSR3 was expressed in
C. reinhardtii under the control of heat shock protein 70/Rubisco small
chain 2 promoter in npq4 lhscr1 background that lacks all LHCSR genes.
7
Algal Research 66 (2022) 102810
This fine tuning and precise gene editing approach improved NPQ
photosynthetic capacity (5 %), and higher biomass productivity in
C. reinhardtii. Lately, the amplitude of rapidly reversible NPQ (energy
dependent quenching-qE) was assessed with high light, blue light and
UV light by regulating the expression of LHCSR and PSBS genes in
C. reinhardtii, and showed that SPA1 and CUL4 are the two putative
components of conserved E3 ubiquitin ligase complex, and its regulation
depends on a transcription factor CrCO [58,59].
5.3. Increasing resistance to oxidative damage
On the other side, in order to prevent photo-oxidative damage caused
by ROS formation, photosynthetic organisms have transformed direct
and indirect cellular mechanisms for recognition of high light intensity
and also accustomed to changes in the environment. During high light
stress condition, elicits the formation of ROS like singlet oxygen (1O2),
superoxide (O−2 ), hydrogen peroxide (H2O2) and hydroxyl radicals,
which causes lipid oxidation, chlorophyll photo bleaching, protein
degradation, DNA damage and reduction in photosynthetic efficiency
[60]. Although, light is potential energy source for most algae, excessive
light leads to multiple depression in photosynthetic systems including
PSII, which caused oxidation (H2O) and reduction (Plastoquinone), and
bind with Chl pair, P680. Several thylakoid-bound antioxidants in the
photosynthetic apparatus of algae, play a significant role in dampening
lipid peroxidation and quenching 1O2. Subsequently investigation
identified that, 1O2 generation in the reaction center of PSII and 3Chl* in
LHC were quenched by β-carotene and xanthophylls. Meanwhile, ROS
scavenging mechanisms and protective function of antioxidants like
ascorbate, glutathione, tocopherol, carotenoids, and related redox pro­
teins and enzymes such as thioredoxin, glutaredoxin, peroxiredoxin,
catalase, ascorbate peroxidase and glutathione peroxidase have been
P. Priyadharsini et al.
reported in plants and algae. Overproduction of CrDHAR1 (Chlamydo­
monas v5.5:Cre10.g456750, glutathione dehydrogenase) in
C. reinhardtii, showed tolerance to oxidative stress, low H2O2 produc­
tion, lipid peroxidation, maintained high Chl contents under high light
illumination [61]. The glutathione reductase, ascorbate peroxidase,
DHA content, AsA pool size and AsA:DHA ratio increased in CrDHAR1-
Knockdown amiRNA lines. An essential role of α-tocopherols in dimin­
ishing photo-oxidation of PSII and its reaction center D1 protein under
low and high light intensities was investigated in C. reinhardtii, besides
that diphenylamines, a chemical quencher of 1O2 protects both PSII and
D1 protein. Overproduction of tocopherol was greatly achieved in
C. reinhardtii with the gene encoding for homogentisate phytyltransfer­
ase vitamin E2 (VTE2), showed 11fold raise in total tocopherol content.
Meanwhile, overexpression of this VTE2 gene in double mutant npq1
lor1 revealed higher efficiency in PSII, and increased resistance to
oxidative stresses [62,63]. Similarly, phosphoprotein SAK1 from
cytosol, inducted and phosphorylated leading to singlet oxygen accli­
mation via retrograde signaling pathway from chloroplast to nucleus in
C. reinhardtii was noted [64]. However, signaling pathways, exact
mechanism that elicit antioxidant defenses under different stress con­
ditions remain enigmas.
5.4. Increased CO2 assimilation routes
Over the past few decades, phycologist have sought attention in
customizing microalgal respiratory pathways, thereby amplify the effi­
ciency of photosynthesis by replacing efficient enzymes including
rubisco, carboxylases, and other complementary pathways linked with
CO2 fixation and Calvin cycle. Rubisco was replaced with three different
carboxylating enzymes like isomerase, a biotin dependent carboxylase
and hydrolase, maintaining Calvin cycle largely intact [65]. In addition
to this, 6-phosphogluconate dehydrogenase (6PGDH) is the best alter­
nate for rubisco, since 6PGDH is a reversible reductive carboxylase with
higher kinetics, Kcat/KM values, increased carbon fixation rate and yield
[66]. Naturally available six carbon fixation pathways are Calvin cycle
(1948) reductive TCA (rTCA pathway, 1966), Wood-Ljungdahl pathway
(WL pathway, 1972), dicarboxylate/4-hydroxybutyrare cycle (Di-4HB
cycle, 2007), 3-hydroxypropionate bicycle (3HP bicycle, 1993), and 3-
hydroxypropinate-4-hydroxybutyrate cycle (3HP-4HB cycle, 2008).
However, Calvin cycle, 3HP and 3HP-4HB cycles involved aerobic
conditions, where the rTCA, WL and Di-4HB reacts under anaerobic
conditions. On the other hand, synthetic pathway can be envisioned in
order to offer tailor made metabolic clues for CO2 fixation [67]. One of
prominent examples is malonyl-coenzyme A (CoA)-oxaloacetate-glyox­
ylate (MOG) pathways, having catalytically fast phosphoenol pyruvate
carboxylase enzymes, which converts acetyl-CoA to malonyl-CoA under
anaerobic and requires only less ATPs. Another group of synthetic CO2
fixing Crotonyl-CoA/ethylmlonyl-CoA/hydroxybutyryl-CoA (CETCH)
cycle, a complex reaction network that converts CO2 into organic mol­
ecules via efficiently active crotonyl-CoA carboxylase/reductase and it
was drafted and metabolically reconstructed with several enzymes from
nine different organisms [68,69]. Similar synthetic pathway called
“malyl-CoA-glycerate (MCG)” was designed in Synechococcus elongates
PCC7942, capable of converting C3 metabolite to two acetyl-CoA, by
supplementing one more CO2 equivalent thereby assimilating glyoxylate
without net carbon loss [70].
Besides these pathways, recently reported complementary cycles can
also be incorporate to trigger the carbon assimilation instead of its
replacement. Formate assimilation pathway, where formate oxidized to
CO2 by formate dehydrogenase (FDH). Through this path, formate is
being transformed into formy-tetrahydrofolate (THF), which further
reduced to methylene-THF, followed by transfer of C1-moiety to glycine
to generate serine [71]. Quite recently, modular engineering approach
with selection strategies were adopted to substitute ribulose mono­
phosphate pathway for methanol assimilation and the reductive glycine
pathway for formate assimilation. This coupled metabolic engineering,
8
Algal Research 66 (2022) 102810
will provide information for precise rewiring algal metabolism for a
novel phenotype with robust CO2 assimilation [72]. Another typical
approaches to elevate the efficiency of CO2 fixation and biomass pro­
duction was NADPH consumption pathway, where genetically engi­
neered Synechocystis sp. PCC 6803 showed additional NADPH
consumption [73]. Seminal synergetic CO2 fixing pathway was carried
out in S. elongatus that significantly improves malate production and
CO2 fixation rate with simultaneous balancing of ATPs between
carboxylation reaction in central metabolic pathway and rubisco shunt
in the carbon fixation pathway [74].
6. Concluding remarks and future directions
Carbon dioxide (CO2) can be utilized as a carbon source by all
photosynthetic microorganisms. Especially in cultivation of microalgae,
CO2 does not act as a rate limiting factor when compared to other fac­
tors. Effect of light on indoor cultivation can be optimized by utilizing
inexpensive LED lights with proper wavelength. The material used for
reactor fabrication also plays a vital role in light penetration inside the
reactor, which helps in photosynthesis process. CO2 delivery and fixa­
tion depends upon the size of air bubbles inside the reactor. Overcoming
the defects in raceway ponds and price of PBRs, hybrid models are
designed and experimented. American research space organization,
National Aeronautics and Space Administration (NASA) has developed
hybrid reactor (Offshore Membrane Enclosures for Growing Algae
(OMEGA)) for higher algae biomass yield. This system facilitates higher
algae cultivation with lesser land utilization, wastewater usage and flue
gas sequestration. Wastewater and sea water utilization for algae culti­
vation in reactor helps in achieving better water footprints.
Carbon dioxide sequestration and utilization was enhanced through
usage of genetically modified organisms (Algae). Mainly in gene editing,
most important factors are mutations with smaller antenna size and NPQ
activity with self-shading effects. Genetically engineering of plants un­
dergoes numerous directives and geographical location also an impor­
tant factor holds the drawback on plants. Integrated methods like algae-
based carbon dioxide sequestration provides economical and environ­
mentally friendly approaches. Improved CO2 sequestration and utiliza­
tion with latest knowledge and engineering tools helps in achieving
enhanced biomass productivity with greater CO2 sequestration. Micro­
algae comprises numerous strain diversity, till now very few strains are
studied and reported. Further research on other algal strains needs to be
explored for sustainable development.
CRediT authorship contribution statement
P. Priyadharsini: Writing - original draft, Methodology.
N. Nirmala: Writing - original draft, Methodology.
Baskran: Writing - original draft.
P. SundarRajan: Review & editing.
K. P. Gopinath: Conceptualization, Data curation.
J. Arun: Project administration, Supervision.
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
Data will be made available on request.
Acknowledgement
Author's wish to thank Sathyabama Institute of Science and Tech­
nology for the support and Freepik for the source of diagram.
P. Priyadharsini et al. Algal Research 66 (2022) 102810

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