Intro Part I

Exponential human growth brings with it an increasing demand of resources, and current production
systems are not expected to cope with the immediate future demands (SOURCE). A world population of
over 10 billion people by 2050 will put unmanageable pressure on natural resources and push global
ecological change to its limits (SOURCE). Facing this scenario implies the shift towards a biobased and
greener economy, in which microalgae stand as a promising feedstock for a wide range of products and
processes (Grama et al., 2016; Kumar et al., 2020; Zhan et al., 2017). Microalgae are unicellular
photosynthetic microorganisms that can be found in all kinds of environmental conditions (Khan et al.,
2018; Zhan et al., 2017). They produce a variety of carbon compounds that can be utilized for many
different purposes, such as biofuels and biopolymers, health supplements and pharmaceuticals,
cosmetics, feed and human food (Alam et al., 2020; Khan et al., 2018; Kumar et al., 2020). Considering
their potential, algal products are expected to have a market of more than 3.4 billion USD by 2025
(Kumar et al., 2020).
These microorganisms grow on light and CO 2 with photosynthetic efficiencies up to 50% higher than
those of terrestrial plants and with an astonishing inorganic carbon fixation capacity, accounting for 40%
of global CO2 sequestration (Santos et al., 2011; Shahid et al., 2020; Zhan et al., 2017). Furthermore, they
do not consume arable land nor potable water and can be grown on wastewater, using inorganic
contaminants as nutrients (Shahid et al., 2020; Zhan et al., 2017). Their production is not subjected to
seasonal variation and can be harvested daily (Daneshvar et al., 2021), thus outcompeting terrestrial
plants in all aspects.
The most common large scale cultivation strategy for microalgae is photoautotrophy (hereafter referred
to as autotrophy, where light acts as the energy source and CO 2 is supplied as carbon source (Chew et al.,
2018; Pang et al., 2019) . Under this regime, light-dependency of growth hinders productivity in various
ways. Low light intensities will translate in a light-limited culture, while high light intensities can trigger
photo-inhibition and photo-oxidation mechanisms, thus damaging the photosynthetic machinery of the
cells (Daneshvar et al., 2021; Das, 2016; Santos et al., 2011). Shading effects are also a key factor
controlling growth rate, since cells distribution reduces light availability at different depths of the light
pathway (Daneshvar et al., 2021; Das, 2016; H. Wang et al., 2020). Concerning carbon supply,
atmospheric CO2 concentration (0.04% v/v) is too low to ensure a proper gas-liquid mass transfer. To
tackle this, CO2-enriched air is needed to prevent carbon depletion in the culture, increasing operational
costs and causing carbon losses to the atmosphere (Abiusi et al., 2020a; Acién et al., 2012; Santos et al.,
2011; Zhan et al., 2017). Considering all of these limitations, autotrophically produced microalgae do not
achieve the productivities needed to make the process economically feasible for industrial scale (Acién et
al., 2012; Acién Fernández et al., 2019; Das, 2016; Deschênes et al., 2015; Santos et al., 2011).
As an alternative to enhance biomass production rates and culture densities, some microalgae are grown
under strict chemoheterotrophic metabolism (hereinafter referred to as heterotrophy) in which no light
is needed and an organic carbon compound is used as both energy and carbon sources (Daneshvar et al.,
2021; Klamczynska & Mooney, 2017; J. Wang et al., 2014). This strategy, as it overcomes illumination-
related problems, allows for microalgae cultivation in conventional industrial scale fermenters and at
high cell densities, reducing the costs of both upstream and downstream processes (Hu et al., 2018;
Venkata Mohan et al., 2015; Zhan et al., 2017). Nevertheless, the number of species that are able to
thrive in darkness is very limited; and even if they can, heterotrophically cultivated cells may exhibit
reduced content of pigments and other biomolecules of interest (Barone et al., 2020; Bumbak et al.,
2011; Hu et al., 2018; Perez-Garcia et al., 2011; Wan et al., 2016).
Mixotrophic cultivation has been pointed out as the strategy capable of solving the major disadvantages
encountered by the beforementioned methods (Deschênes et al., 2015; Perez-Garcia & Bashan, 2015; J.
Wang et al., 2014). Under mixotrophic metabolism microalgae are able to use light and CO 2 as normally
done with the photosynthetic machinery, while simultaneously using organic carbon as both energy and
carbon sources. This concurrent performance of autotrophy and heterotrophy is well-extended among
species, and has been reported to yield considerable higher productivities than autotrophy (Abiusi et al.,
2020b; Pang et al., 2019; H. Wang et al., 2020; Zhan et al., 2017). Additionally, while growing
mixotrophically CO2 fixation is still occurring, and the positive environmental impact of microalgae
cultivation is maintained (Deschênes et al., 2015). Nevertheless, the development of a feasible large
scale production process still has to address some important issues.
One of these key concerns is related to gas-liquid exchange during cultivation. Since CO 2 is one of the
essential reactants for autotrophic metabolism its concentration in the liquid has to be enough to
satisfy cells requirements at any moment during illumination of the culture (Prokop et al., 2015). On
the other hand, oxygen, which is a product of0 photosynthetic activity, has a harmful effect on the
microorganisms when its concentration builds up in the liquid, principally via the processes of
photorespiration and photoinhibition (Kazbar et al., 2019; Moroney et al., 2013; Perez-Garcia &
Bashan, 2015; Sousa et al., 2013). Because of these two molecules, the gas-liquid mass transfer is one
of the most important features in the design and operation of any system for microalgae cultivation
(Das, 2016; Molina Grima et al., 2000; Ugwu et al., 2008). In open large scale cultivation systems
(raceway ponds), gas exchange is not optimized and CO2 dissipates into the atmosphere, while O2 is not
efficiently removed (Gupta et al., 2015; Prokop et al., 2015). Closed systems known as
photobioreactors (PBR) offer a more refined control of gas exchange, but to enhance mass transfer
between both phases large amounts of energy are needed (Ruiz et al., 2016). As a result,
photobioreactors dimensions and design always have to take into account the need for gas-liquid mass
transfer, and the related intense aeration accounts for a considerable part of the operational costs
(Acién Fernández et al., 2019; Janssen et al., 2003; Venkata Mohan et al., 2015).
As an improvement of mixotrophy, a new cultivation strategy known as oxygen balanced mixotrophy

(OBM) has recently been developed by Abiusi and colleagues (Abiusi et al., 2020a). The methodology
aims at enhancing the internal recirculation within the microalgal cell of O2of O2 in the microalgal cell,, in
such a way that no gas-liquid mass transfer is needed in the culture as long as autotrophic metabolism is
occurring (Figure 1; Abiusi et al., 2020b, 2020a). The oxygen production rate from photosynthesis is
coupled to the heterotrophic oxygen consumption rate, so the O 2 produced during photosynthesis does
not accumulate since it is consumed for cellular respiration, and no external oxygen input is needed in
the culture. This approach also stimulates the reutilization of the CO 2 produced during respiration as the
carbon source for autotrophic growth, eliminating the need of CO 2 supply. Still, a small percentage of the
CO2 produced during heterotrophy is released, due to stoichiometric differences between the two
metabolic pathways. At an operational level, the gas recirculation is achieved by changing the organic
carbon source supply rate based on the measured variations in dissolved oxygen (OD) in the medium,
with the goal of keeping the OD stable. This strategy was first successfully applied for cultivation of
Chlorella sorokiniana. Results showed higher biomass productivity and concentration compared to an
autotrophic culture, and much higher yield of biomass on substrate than the one reported for a strict
heterotrophic culture (Abiusi et al., 2020a). Even more, photosynthetic machinery did not suffer any
apparent detriment derived from the presence of an organic carbon source, in contrast with data
reported in previous occasions (Grama et al., 2016; Perez-Garcia & Bashan, 2015; Zhan et al., 2017).
Figure 1. Schematic overview of a microalgae under oxygen balanced mixotrophic metabolism.
Another important factor to take into account, specifically when organic compounds are present in the
medium as it is the case of mixotrophic cultivation, is the risk of contamination (Abiusi et al., 2020a;
Daneshvar et al., 2021; Deschênes et al., 2015; Hu et al., 2018). To prevent the growth of undesired
microorganisms large efforts and resources are put on proper sterilization, challenging the upscaling of
the strategy (Di Caprio, 2020). The use of extremophilic species is considered as a possible solution to
this problem. These species are able to grow under extreme conditions that act as selective environment
and guarantee that the culture remains axenic (Malavasi et al., 2020; Sydney et al., 2019).
Among this group, Galdieria sulphuraria is currently gaining attention as a suitable organism for a variety
of biotechnological applications (Čížková et al., 2019; Sydney et al., 2019). G. sulphuraria is a
polyextremophilic red microalgae that grows at low pH levels (0-4), high temperatures of up to 57 °C,
and stands osmotic pressures of up to 400 g·L-1 (Graverholt & Eriksen, 2007; Sydney et al., 2019). It
synthesizes the pigment-protein complex phycocyanin, currently produced from Spirulina at industrial
level (Kuddus et al., 2013; Sydney et al., 2019; Wan et al., 2016). Phycocyanin can be used as colorant in
both food and cosmetics industries, it exhibits many interesting therapeutic properties and it is also
commercialized as fluorescent marker in biomedical research and diagnosis (Čížková et al., 2019; Pagels
et al., 2019; Abiusi & Moñino, submitted). Comparison studies have demonstrated that G. sulphuraria’s
phycocyanin tolerates hotter and more acidic conditions than the one produced from Spirulina (Wan et
al., 2016; Abiusi & Moñino, submitted). G. sulphuraria has also reported higher phycocyanin productions
than Spirulina (Graverholt & Eriksen, 2007; Wan et al., 2016). All this considered, this extremophilic
species seems to have the capacities to overcome Spirulina as the main phycocyanin producer; , but. iIt
also has other interesting potential applications, such as wastewater treatment (Henkanatte-Gedera et
al., 2017) or rare earth elements recovery (Čížková et al., 2019).
Very recently, the strain Galdieria sulphuraria 064 has proven to effectively grow under OBM conditions
and to maintain the phycocyanin content unaltered in comparison to an autotrophic culture (Abiusi et
al., submitted; Abiusi & Moñino, submitted). Moreover, the resulting protein content from OBM
cultivation was considerably high (≈60% w/w) and compliiled with the FAO requirements of essential
aminoacids (Abiusi & Moñino, submitted). This information suggests that G. sulphuraria grown under
OBM conditions could be a valuable source of protein for human diet.

Intro Part I

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Exponential human growth brings with it an increasing demand of resources, and current production

As an improvement of mixotrophy, a new cultivation strategy known as oxygen balanced mixotrophy

Figure 1. Schematic overview of a microalgae under oxygen balanced mixotrophic metabolism.

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