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Chapter 40

Disruptive technologies to improve


the performance of microalgae cultures
Rosangela Rodrigues Dias, Mariany Costa Deprá, Leila Queiroz Zepka, and Eduardo Jacob-Lopes
Bioprocess Intensification Group, Federal University of Santa Maria, UFSM, Santa Maria, RS, Brazil

1 Introduction to the state of the art Today, the relevance of microalgae as a rich source of
of microalgae biotechnology feedstock for natural products is increasing. The size of
the global microalgae-based product market was estimated
Microalgae (including cyanobacteria) are a distinct group of at USD 4.7 billion in 2021 and is expected to reach
microscopic organisms with around 30,000 documented approximately USD 6.4 billion in 2026. The North
species. However, while there may be thousands of species American microalgae market is considered the most
consistently cataloged, it is worth mentioning that only a mature. This can be attributed to the expansion of com-
few hundred are investigated as to the physiological and mercial production facilities that are driving the market
biochemical characteristics and only a handful of them in the region. Furthermore, there is an increase in product
are actually cultivated commercially for the production of innovation and exploration of new applications of micro-
microalgae products (Mobin and Alam, 2017). algae which unfortunately is not witnessed in underde-
In the last years, the biotechnological potential of micro- veloped countries due to a lack of R&D activities
algae has awoken global interest fundamentally because (MarketsandMarkets, 2022).
they offer a unique platform for the production of a series However, regardless of this, it is important to highlight
of products such as transport fuels, food, feed, fertilizers, that extensive applications of microalgae in various sectors,
pharmaceuticals, and personal care products. Of impor- such as energy, nutrition, and cosmetics, are one of the main
tance, although commercial production of microalgae is factors that should drive the growth of the microalgae market
considered recent, the consumption of microalgae espe- in the coming years. Noteworthily, microalgae have
cially as food has been a common practice for centuries attractive commercial aspects such as rapid growth in relation
(Markou et al., 2021). to terrestrial cultures, cellular structures that allow easier
These microorganisms that include prokaryotic and extraction of products, tolerance to several environmental
eukaryotic beings, autotrophs, and many capable of devel- factors, and being able to be cultivated in small spaces and
oping heterotrophically, provide basic nutrition that regions unsuitable for agriculture (Fabris et al., 2020).
includes carbohydrates, lipids, and proteins and can also In the state of the art, although microalgae are consid-
bring additional health benefits. This is because they are erably established in the market, it is indisputable that the
sources of a variety of compounds that have biological upstream and downstream processes have a relatively high
activities such as antioxidant, anti-inflammatory, anti- cost. Besides that, despite having the potential for several
cancer, and antiviral (Dewi et al., 2018). biotechnological applications, they are used today for a
Historically, large-scale commercial microalgae small and embarrassing number of industrial applications
culture began around 1960 with the culture of Chlorella (Deprá et al., 2020). However, the development and use of
sp. followed by Arthrospira sp. which began to be culti- emerging technologies can leverage the promising capabil-
vated commercially in the 1970s. Whole dried biomass ities of these microorganisms. Under this horizon, the aim
was the main product that the industry was targeting. Later of the chapter is to highlight how disruptive technologies
arose was the production of b-carotene and astaxanthin such as genetic engineering and the Industrial Internet of
from Dunaliella sp. and Haematococcus sp. and, more Things can improve the performance of microalgae cul-
recently, the production of polyunsaturated fatty acids tures and promote their advancement to become a platform
(Dias et al., 2020). for sustainable products and next generation.

Handbook of Food and Feed from Microalgae. https://doi.org/10.1016/B978-0-323-99196-4.00034-6


Copyright © 2023 Elsevier Inc. All rights reserved. 529
530 PART V Challenges and recent advances

2 Disruptive technologies to improve and basic methods such as (i) DNA transformation,
the performance of microalgae cultures (ii) functional promoters, terminators, and other genetic ele-
ments, and (iii) selection markers (Korpys-Woźniak et al.,
2.1 Genetic engineering (GE) 2022). In addition, another fundamental criterion for the
excellence of genetic manipulation is the establishment of
Manipulating the cellular metabolism of microalgae is a
an axenic culture. Thus, once this complex interconnected
long-standing challenge in microalgal biotechnology. In
system is defined, these indicators can be combined and,
fact, it is not new that massive efforts have been made by
therefore, will result in a comprehensive and reliable
scientists and researchers to try to optimize these biological
cloning system (Kselı́ková et al., 2021).
processes (Thanigaivel et al., 2022). This is because, beyond
intrinsic concerns related to the operational parameters of
cultivation and high production costs, problems of a bio-
logical nature involving cell productivity increase, as well
2.1.1 Gene transformation and selection
as the synthesis induction of specific metabolites of com- methods
mercial interest, have been investigated under the genetic Gene transfer has been possible thanks to the genetic manip-
and metabolic engineering perspective (Brar et al., 2021; ulation of cells, which use direct or indirect methods of
Kang et al., 2021). transformation. Direct gene transfer methods use physical
Genetic engineering can be understood as one of the or chemical processes that cause changes in the cell walls
main pillars of biotechnology. Genetic engineering encom- and membranes, facilitating the introduction of exogenous
passes a techniques range manipulating nucleic acids DNA. Several direct methods have been proposed, varying
(Box 1), generally through their recombination, with the in their efficiency and practicality. Among them, the direct
objective of synthesizing improved microorganisms, aiming methods that provide the largest number of transformed
at the improvement or genetic structuring of a given species species are agitation with glass spheres, electroporation,
according to scientific needs (Sharma et al., 2022). and particle acceleration/bombing (Fig. 1A). In contrast,
However, genetic engineering—applied to microalgae— the indirect method is one in which a vector, such as
has only received more attention in recent years due to Agrobacterium-based, is used to mediate gene transfer
the improvement of its tools, resulting in increasingly acces- (Fig. 1B) (Mosey et al., 2021).
sible methods, since the identified genomes can be edited Evidently, any technic has its own pros and cons based
according to the required needs much more accurately than on efficiency, integration, or transgene stability. However,
before (Muthukrishnan, 2022). the glass bead agitation method has been presented as a
When applied specifically to biotechnology, genetic pattern protocol for DNA transfer from microalgae such
engineering requires the development of different tools as Chlamydomonas reinhardtii, Cyanidioschyzon merolae,
Chlorella vulgaris, and Dunaliella salina due to its low cost
and operational simplification (Shi et al., 2021). Fur-
thermore, this method can be employed for species whose
BOX 1 Basic characteristics of nucleic acids.
By definition, nucleic acids are intracellular macromolecules
classified in two forms—deoxyribonucleic acid (DNA) and TABLE 1 Nucleic acid differentiation parameters.
ribonucleic acid (RNA)—which have the chemical consti-
tution and distinct biological functions. DNA is considered Parameters DNA RNA
the most important since it constitutes the genome of living
Function Store and perpetuate Transcribe the
beings, storing all the genetic information (genetic code) the genetic code genetic code
essential for their existence. Other relevant information com-
prises the varied types of DNA: genomic, mitochondrial, Types Genomic, plasmid, Ribosomal,
chloroplast, and plasmid (Table 1). In microalgal microor- mitochondrial, and messenger,
chloroplast transporter
ganisms, genetic manipulation usually takes place in chloro-
plasts. Unlike DNA, RNA can be observed in the nucleus and Number of 2 1
cytoplasm of the cell. They are molecules much smaller than structural
DNA and less stable. The types of RNA found are ribosomal, chains
messenger, and transporter. Its function consists of protein Nitrogenous Adenine, thymine, Adenine, uracil,
synthesis, which is the final compound of the expression of bases guanine, and cytosine guanine, and
a genetic character stored in the DNA. Therefore, it can be cytosine
said that the structural and enzymatic functions are attributed
Base sugar Deoxyribose Ribose
to RNA (Ableitner, 2022).
Disruptive technologies to improve the performance of microalgae cultures Chapter 40 531

FIG. 1 Genetic methods for DNA transformation of microalgal microorganisms. (A) Direct gene transfer methods and (B) indirect gene transfer
methods.

cell wall is unsuitable or when a chemical or enzymatically 2.1.2 Gene editing methods—Gene
removing pretreatment is applied. expression alteration
Likewise, particle bombardment/acceleration transfor- Modifying gene expression is a common task assigned to
mation—also recognized as biolistics—can be applied to genetic engineering. Currently, there are two ways to
transform microalgae that contain resistant cell walls. Tech- achieve it either through (i) knock-outs, the process of
nically, this method is based on accelerated microprojectiles completely removing a gene from an organism or (ii)
of non-reactive metal (gold or tungsten), coated with DNA, knock-ins, the process of inserting a new gene into an
being fired and collided with the target cells. As a result, this
organism (Kselı́ková et al., 2021).
impact allows the particles to penetrate cells and deliver the Given this foundation, it is possible to consider that
foreign DNA (Fajardo et al., 2020). While this technique is genome editing techniques are nothing more than processes
successful in disrupting the microalgae cell walls, it is of adding, deleting, and correcting genes, after rewiring the
restricted due to the high equipment cost and specific con- metabolic pathways, beyond performing other highly tar-
figurations required for its procedure. However, for chloro- geted genomic modifications. However, it is known that
plast transformations, the particle bombardment method is DNA-directed changes start from the generation of
more efficient than glass bead agitation (Barati et al., 2021). nuclease-induced double-strand breaks (DSBs), which leads
Conversely, the action mechanism of the electroporation
to stimulation of highly efficient recombination mecha-
method uses electrodes to reproduce a voltage differential nisms of cellular DNA (Li et al., 2020). DSBs of
across the cell membrane. This electrostatic difference nuclease-induced DNA can be reconditioned by one of
results in a temporary disturbance of the phospholipid the two major arrangements that happen in almost all cell
bilayer, allowing molecules to pass into the cell (Singh types and organisms: homology-directed repair (HDR)
et al., 2022). According to the literature, electroporation and non-homologous end-joining (NHEJ), providing tar-
provided cell wall disrupture for microalgal and cyanobac- geted integration or gene disruptions, respectively (Khan
teria transformations, such as C. reinhardtii and Anabaena et al., 2022).
(Gutierrez and Lauersen, 2021).
Therefore, the first gene editing techniques were based
Additionally, the indirect transformation method on RNA-mediated silencing. RNA interference (RNAi),
mediated by Agrobacterium sp. represents one of the most the most widely used technique, is an evolutionarily con-
cutting-edge methods for stable integration of foreign genes served post-transcriptional process that exploits cellular
into host cells. In fact, this technique exploits bacterial path- mechanisms involved in defense against viruses and trans-
ogenicity through its ability to carry over foreign genes posons and the regulation of endogenous genes ( Jeon et al.,
using the T-DNA region of their tumor-inducing plasmid 2017; Fajardo et al., 2020). It is activated by double-
(Ti plasmid) into a host genome (Norzagaray-Valenzuela stranded RNA and leads to gene silencing. However, more
et al., 2018). Among its significant advantages, the decrease
recently, gene editing tools such as zinc-finger nucleases
in the number of transgene copies, DNA rearrangements, (ZFNs), meganucleases (MNs), transcriptional activator-
and transgene silencing are among the highlights of the like effector nucleases (TALEN), and clustered regularly
technique (Mini et al., 2018). However, process control interspaced short palindromic repeats (CRISPR/Cas9)
parameters such as co-culture time, temperature, pH, and emerged as the most successful tools for gene editing
microalgae target species have been indicated as key factors (Zhang et al., 2019). In short, these new tools have the same
for the success of microalgal genetic modification.
532 PART V Challenges and recent advances

FIG. 2 Principles and mechanisms of gene editing. The break mechanisms shown are the ZFN (zinc finger nucleases), TALEN (transcription activator-
like effector nucleases), and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats). NHEJ (non-homologous end-joining repair) and
HDR (homologous-directed repair) are the repair mechanisms.

mechanism of action, that is, they are capable of breaking the internal connection between gene expression and the
the double strand in the DNA sequence (Fig. 2). Besides, phenomena of cellular life. More specifically in the micro-
their applicability depends on economic parameters, opera- algal biotechnology field, transcriptomics is adopted for
tional complexity, and the ability to simultaneously cause concrete identification or detection or to expand knowledge
different editions (Lin et al., 2019). and understanding of specific metabolic pathways or bio-
logical processes (Brar et al., 2021; Muthukrishnan, 2022).
Additionally, proteomic studies focus on exploring the
2.1.3 Multi-omics technology characteristics of cellular proteins. This investigation
Omic technologies aim to provide a broad view of the inner includes protein expression levels, post-translational modi-
workings of cells. When applied to microalgal microor- fications, protein interactions, and other protein functions at
ganisms, they aim to understand molecular integrity. Gen- the cellular level (Gu et al., 2021). Technically, proteomic
erally, microalgal omic studies focus on genomics, technology is segmented into two broad areas: protein sep-
transcriptomics, proteomics, and metabolomics (Fig. 3) aration and identification. These subdivisions are used in
(Maghembe et al., 2020). order to promote the protein profile evaluation of the subcel-
As seen in Fig. 3, recent advances in omic technology lular compartments or to investigate the microalgal cellular
focus on four distinct areas, where the focus of cellular study metabolism under stress conditions.
and investigation differs according to the mechanism of More recently, among other omic technologies, metabo-
action. Namely, genomics is a discipline that investigates lomics has emerged. This omic segment refers to the inves-
the composition of biological genomes, precise structure, tigation of low-molecular-weight biomolecules—such as
and relationship of each gene, as well as the regulation of amino acids, peptides, carbohydrates, and lipids—released
expression. Its main purpose is to characterize and quantify during cellular metabolic processes as the final response
all genes of a microorganism, as well as to identify their of the biological system against genetic and environmental
function in the cellular metabolic system. It is customary changes (Kato et al., 2022). Currently, metabolomics is sup-
to divide genomics into structural and functional. These ported by state-of-the-art technologies and relies on gas
functions are directly associated with the part of the cellular chromatography-mass spectrometry (GC-MS), liquid
DNA (Pandey et al., 2022). During this, the transcriptome chromatography-mass spectrometry (LC-MS), and nuclear
delves into the process of gene transcription and tran- magnetic resonance (NMR) techniques as the pillars of your
scription regulation in cells in an integral way. This tech- analyses (Ye et al., 2022). In the microalgal application,
nology, in turn, is capable of quantifying the level of metabolomics is attributed to the quantification and identi-
differential gene expression in cells under different condi- fication of secondary metabolites with significant com-
tions. Through these analyses, it is possible to understand mercial value directed to pharmaceutical and food science.
Disruptive technologies to improve the performance of microalgae cultures Chapter 40 533

FIG. 3 Strategies for multi-omics profiling applied to microalgae cells.

As noted throughout the section, metabolic engineering people, processes, and other things. It is likely that this is
techniques—although they have advanced in recent times— one of the technology trends that will cause more disruption
are still too complex to comprehensively achieve. In fact, it in the coming years (Nord et al., 2019).
is probable that in the coming years, the methodological In parallel, the Industrial Internet of Things (IIoT) as a
complexity will provide space for more direct and faster subset of the IoT is a vital technology of the fourth industrial
analyses, that is, without so many adjustments in the meta- revolution. This technology is revolutionizing the way
bolic pathways of interest (Bharathiraja et al., 2022). industrial organizations are executed. That is because it
With the advent of systems and synthetic biology tools, it includes monitoring applications and approaches to self-
is expected that the metabolic engineering of microorganisms organizing production systems that require little or no
to produce increasingly complex compounds will be done in human intervention. The technology in question is an evo-
a more targeted and fast way, that is, without the need for so lution of the IoT and paves the way for more efficient and
many adjustments of the metabolic pathways (endogenous or sustainable production (Sisinni et al., 2018).
heterologous) of interest. However, genetic and metabolic Of note, increased production performance through
genetic engineering has proved to be an important research intelligent and remote management has been one of the
tool and has increasingly contributed to advancing disruptive main reasons for the adoption of IIoT by companies pro-
technologies applied to microalgal processes (Lu et al., ducing consumer goods. In particular, the great promise
2022). Advances in the literature in the last decade have given offered by the IIoT has awakened the interest of many
rise to a wide range of publications involving genetic engi- researchers who aim to promote the microalgae industry.
neering and microalgae (Table 2). Research has generally The microalgae industry is known to face many bottlenecks
focused on genetically modifying algae for optimized fuel that need to be resolved. But technology nodes can be untied
production, i.e., improvements in lipid yields, starch conver- using IIoT technologies. The application of IIoT in the
sions, and production of green energy such as biohydrogen microalgae industry could be the definitive key for it to gain
(Banerjee et al., 2020; Fayyaz et al., 2020). However, due ground worldwide (Wang et al., 2021).
to nutritional and commercial relevance, some studies are According to recent studies, IIoT can really promote
emerging on the genetic modification of microalgae to microalgae-based process automation. The technology can
optimize the production of isoprenoids, such as astaxanthin be present in any part of the process that requires auto-
and zeaxanthin (Sproles et al., 2021). mation, capture, transfer, and processing of information.
In a few words, adapted processes capable of adjusting to
changes in the process itself can be created. The result is
2.2 Industrial Internet of Things (IIoT)
automated processes that, besides providing lower operating
The Internet of Things (IoT) is a concept that basically costs, can allow real-time monitoring of microalgae growth
describes the connection of physical objects to the Internet. kinetics. Furthermore, economically unfeasible products
The central idea is that the physical and digital worlds such as microalgae biofuels may finally become feasible
become one. This emerging technology can combine a using this emerging technology (Fabris et al., 2020).
myriad of information capture devices with the Internet to Sensors as indispensable elements of IIoT can be used in
form a ubiquitous network capable of interconnecting the upstream and downstream processing of microalgae for
534 PART V Challenges and recent advances

TABLE 2 Genetic modification in microalgal cells for biofuels and chemical fine productions.

Strain Modification Modification


microalgae Metabolic alteration method observed References
Biofuels
Chlamydomonas Creation of amiRNAs against CrPEPC Transformation Increased fatty acid Wang et al.
reinhardtii of amiRNA in from 0.086 to (2017)
algae 0.105 g g 1
C. reinhardtii Insertion of BnDGAT2 in C. reinhardtii from Electroporation Increased fatty acid Ahmad
Brassica napus from 0.0298 to et al. (2015)
0.0769 g g 1
C. reinhardtii Knockdown of OEE gene amiRNA coupled Twofold increase in Li et al.
gene knockdown H2 production (2015)
C. reinhardtii Expression of the amylase (arAmyBH) gene Glass-bead- Up (20%) of starch Wang et al.
from Thermotoga neapolitana mediated hydrolysis (0.843 g (2015)
transformation maltotriose from 1 g of
starch)
Chemical fine

Synechococcus Exogenous DNA from Haematococcus Homologous Zeaxanthin yield of Sarnaik


elongatus pluvialis under the control of cpc560 recombination 9 mg/g DCW et al. (2018)
promoter (for BKT) and psbA2 promoter (for strategy using
CrtR-B) plasmids
H. pluvialis Endogenous phytoene desaturase coding Overexpressed in Astaxanthin yields of Galarza
sequence (pds) chloroplast up to 34.3 mg/L et al. (2018)
compared to 18.0 mg/L
in wild-type algae

the management of the facilities. Predictive modeling promising. Recently, Hermadi et al. (2021) attempted to
methods and decision support systems coupled can also develop a system of smart algae ponds using IIoT. Among
help. Sensors to constantly monitor abiotic environmental the highlights is the significant reduction in electricity con-
factors such as temperature, light, and pH and relevant cul- sumption (from 0.5 to less than 0.2 Kw/h). As is well known,
tivation parameters such as biomass concentration have microalgae-based processes are energy-intensive, and
been developed (Giannino et al., 2018; Barbosa et al., reducing consumption positively impacts the environmental
2020). Recently, Esposito et al. (2017) presented an IoT and economic sustainability of these processes (Dias
application for monitoring and controlling microalgae et al., 2022).
growth. The application was coupled with biological Additionally, sensors for monitoring and controlling
modeling for decision support systems. The monitoring harmful and toxic microalgae and in-situ detection and
structure, modeling, and decision support system presented screening have received attention. Hessel and Metfies
were able to produce a 9% increase in productivity. More (2014) developed an automatic monitoring sensor for the
specifically, the structure helped the operator to choose detection of toxic microalgae, which could be significant
the best performance scenario of the culture almost daily in the field of water resource management. Shin et al.
and was able to warn of the proximity of danger to the (2015), in turn, developed a portable fluorescent sensor
cultivation. for microalgae detection on site. Currently, although there
It is noted that culture productivity has not increased are consolidated methods for observation of the phyto-
significantly but could be improved if a huge network of plankton community and detection and quantification of
monitoring and control sensors were implemented simulta- microalgae, the samples need to be transported and carried
neously. About this, stands out that the development of out in laboratories by experienced analysts. This gave way
sensors for microalgae cultivation systems is in its infancy to the sensor development that overcomes this complexity.
and, unfortunately, most in the state of the art does not meet However, there are few studies on the development of
the requirements of large-scale microalgae culture (Wang these sensors and their performance still needs to be
et al., 2021). But, regardless of this, the findings are enhanced.
Disruptive technologies to improve the performance of microalgae cultures Chapter 40 535

Of importance, microrobots and microalgae-based Bharathiraja, B., Iyyappan, J., Gopinath, M., Jayamuthunagai, J., Praveen
sensors are being developed. These microalgae-based Kumar, R., 2022. Transgenicism in algae: challenges in compatibility,
devices are a great promise, but in the status quo, the tech- global scenario and future prospects for next generation biofuel pro-
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Brar, A., Kumar, M., Soni, T., Vivekanand, V., Pareek, N., 2021. Insights
results are hopeful. By way of example, Kashem et al.
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Dias, R.R., Severo, I.A., Deprá, M.C., Maroneze, M.M., Zepka, L.Q.,
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