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Review Not peer-reviewed version
The Microalgae
Chlamydomonas
for
Bioremediation and Bioproduct
Production
Carmen M Bellido-Pedraza , Maria J Torres * , Angel Llamas *
Posted Date: 3 June 2024
doi: 10.20944/preprints202406.0002.v1
Keywords: Microalga;
Chlamydomonas
; bioremediation; wastewater; high-value-added products
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Review
The Microalgae Chlamydomonas for Bioremediation
and Bioproduct Production
Carmen M. Bellido-Pedraza †, María J. Torres † and Ángel Llamas *
Department of Biochemistry and Molecular Biology. Campus de Rabanales and Campus Internacional de
Excelencia Agroalimentario (CeiA3). Edificio Severo Ochoa, University of Córdoba, Spain
* Correspondence: bb2llaza@uco.es; Tel.: +34-957-218352
† These authors contributed equally to the work .
Abstract: The extensive metabolic diversity of microalgae, coupled with their rapid growth rates and cost-
effective production, position these organisms as highly promising resources for a wide range of
biotechnological applications. These characteristics allow microalgae to address crucial needs in both the
agricultural, medical, and industrial sectors. Microalgae are proving to be valuable in various fields, including
the remediation of diverse wastewater types, the production of biofuels and biofertilizers, and the extraction
of various products from their biomass. For decades, the microalga Chlamydomonas has been widely used as a
fundamental research model organism in various areas such as photosynthesis, respiration, sulfur and
phosphorus metabolism, nitrogen metabolism, and flagella synthesis, among others. However, in recent years,
the potential of Chlamydomonas as a biotechnological tool for bioremediation, biofertilization, biomass, and bio-
products production has been increasingly recognized. Bioremediation of wastewater using Chlamydomonas
presents significant potential for sustainable reduction of contaminants and facilitates resource recovery and
valorization of microalgal biomass, offering important economic benefits. Chlamydomonas has also established
itself as a platform for the production of a wide variety of biotechnologically interesting products, such as
different types of biofuels, and high-value-added products. The aim of this review is to achieve a
comprehensive understanding of the potential of Chlamydomonas in these aspects, and to explore their
interrelationship, which would offer significant environmental and biotechnological advantages.
Keywords: Microalga; Chlamydomonas; bioremediation; wastewater; high-value-added products
1. Introduction. Why Microalgae and Why Chlamydomonas?
Microalgae represent a broad array of single-celled, photosynthetic organisms that serve as key
contributors to primary production across our planet [1]. Microalgae can adopt photoautotrophic,
heterotrophic, or mixotrophic modes of life, displaying a spectrum of cell sizes, shapes, and
structures. Responsible for a significant portion of the global carbon capture, microalgae play a
crucial role in supporting ecosystems [2]. Microalgae share a common evolutionary origin that can
be traced back to a primary endosymbiotic event involving a cyanobacterium, which eventually
evolved into the plastid [3]. This process has resulted in the emergence of a wide range of colorful
and metabolically diverse algal groups, such as diatoms and dinoflagellates [4]. Microalgae are
employed in activities such as wastewater treatment [5], biofuel generation [6], animal feed
production [7], and the extraction of high-value-added products [8], among other applications.
Additionally, microalgae show great potential as organisms for enhancing biological carbon
sequestration aimed at mitigating global warming [9]. Consequently, microalgae hold significant
ecological and economic potential.
Chlamydomonas is a microalga that is commonly found in freshwater and saltwater habitats, as
well as in soil and snow. Taxonomically, the genus Chlamydomonas comprises more than 500 species
[10]. Over time, it has evolved into a highly influential model organism, thanks to its numerous
interesting characteristics [11]. Among the Chlamydomonas species, Chlamydomonas reinhardtii is the
most commonly used due to its interesting characteristics. Among these features, C. reinhardtii has
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two flagella, grows well in axenic cultures, exhibits a relatively rapid doubling time of approximately
8-12 hours, and its nuclear, chloroplast, and mitochondrial genomes are sequenced. Additionally, C.
reinhardtii exhibited an exceptional ability to adapt and thrive under nearly all experimental
conditions tested in heterotrophic, phototrophic, and mixotrophic cultivations [12]. Moreover, the
Chlamydomonas Sourcebook [13] provides a thorough overview of essential research areas, historical
background, physiology, and methodologies related to Chlamydomonas. Additionally, the
Chlamydomonas Resource Centre offers a wide range of resources, including biochemical assays,
protocols, plasmids, and a diverse collection of mapped mutant strains. Furthermore, enhancing the
yield of many biotechnological processes involving Chlamydomonas can be achieved through
synergistic interactions with other microorganisms, predominantly bacteria [14].
However, there are still numerous challenges hindering the efficient utilization of
Chlamydomonas biotechnologically in bioremediation and bio-product production. Consequently,
substantial efforts are being directed towards gaining a deeper understanding of the biological
mechanisms relevant to its applications. To the best of our knowledge, there has never been a single
comprehensive review covering all these aspects of Chlamydomonas. Therefore, here we summarize
and categorize these reports with the aim of highlighting the potential of Chlamydomonas to fulfill
these tasks.
2. Wastewater and Advantages of Using Microalgae for Its Bioremediation
Wastewater comprises a diverse mixture of organic and inorganic compounds, as well as
synthetic substances that reflect societal lifestyles and technology. Carbohydrates, fats, sugars, and
amino acids are among the primary contaminants found in wastewater. Indeed, amino acids
constitute three-quarters of the organic carbon in some wastewater [15]. Inorganic constituents found
in wastewater include a variety of substances such as calcium, sodium, magnesium, potassium,
sulfur, arsenic, bicarbonate, heavy metals, nitrates, chlorides, phosphates, and non-metallic salts [16].
Persistent organic pollutants include chlorinated and aromatic compounds, such as polychlorinated
biphenyls, polycyclic aromatic hydrocarbons, and organochlorine pesticides [17]. The composition of
wastewater varies depending on its source. Municipal wastewater is generated from households,
commercial establishments, and institutions. It typically contains organic matter, nutrients,
pathogens, various chemicals from soaps, and detergents [18]. Agricultural wastewater originates
from farming activities and can contain organic matter, pesticides, herbicides, and fertilizers [19].
Industrial wastewater may include a diverse array of industry-specific pollutants, including heavy
metals, organic chemicals, and oils [20]. Each type of wastewater has its own unique characteristics
and requires specific treatment approaches to address its particular contaminants.
As anthropogenic activities increase, resulting in more complex wastewater compositions, it
becomes crucial to develop wastewater treatment procedures that are easy to implement, efficient,
and environmentally friendly. Traditional methods for treating wastewater include physical,
mechanical, chemical, and biological approaches (Figure 1). Physical methods entail processes such
as sedimentation, screening, and skimming, while mechanical methods include filtration techniques
like ceramic membrane and sand filter technology [21]. Chemical methods involve processes such as
neutralization, adsorption, precipitation, disinfection, ion exchange [22]. However, purely physical-
chemical methods have proven ineffective in treating wastewater with complex compositions.
Biological methods for wastewater treatment involve the use of microorganisms that consume
pollutants in the wastewater as food [23]. However, biological wastewater treatment also has various
drawbacks, including high energy consumption, expenses associated with aeration, and challenges
in sludge management. Therefore, the integration of physical-chemical and biological methods is an
effective approach for sustainable wastewater treatment [24].
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Figure 1. The Chlamydomonas-based phycoremediation process for wastewater treatment. The
diagram depicts the three main sources of wastewater. Traditional treatments (physical, mechanical,
and chemical) are shown to be ineffective for treating complex wastewater compositions. These
methods consume high energy and fail to address certain contaminants. Microalgae and macroalgae
play a crucial role in phycoremediation, offering several benefits as indicated. The utilization of
Chlamydomonas in phycoremediation, detailing the main cultivation methods, the employed
mechanisms, and the various compounds that can be bioremediated, is illustrated.
Phycoremediation (where 'phyco' means algae in Greek) is a sustainable and environmentally
friendly approach that utilizes various types of algae, including cyanobacteria, microalgae, and
macroalgae, to remove or extract pollutants from wastewater (Figure 1). Among the benefits of
phycoremediation are the removal of nutrients and xenobiotic substances, the reduction of excess
nutrients from effluent with high organic material, CO2 mitigation, the treatment of effluents with
heavy metal ions, and the monitoring of potentially toxic substances using algae as biosensors [25].
Microalgae have the ability to absorb and break down contaminants through processes such as
biosorption, bioaccumulation, and biotransformation [26]. Phycoremediation not only helps in the
removal of pollutants but also results in the production of algal biomass, which can be utilized for
various valuable products such as food, feed, fertilizers, pharmaceuticals, and biofuels [27]. A wide
range of non-pathogenic algae are utilized for wastewater treatment, such as Chlorella sp., Spirulina
sp., Scenedesmus sp., Nostoc sp., and Oscillatoria sp., [28]. In this review, we will focus on those studies
that use Chlamydomonas in phycoremediation.
3. Microalgae Cultivation Methods
Microalgae cultivation methods are categorized into suspended systems (including open
reactors and closed photoreactors) and attached systems (such as biofilm reactors and encapsulated
microalgae) (Figure 1).
3.1. Open Reactors
Open reactors include lakes and natural ponds, as well as specially designed high-rate algal
ponds (HRAPs) that are tanks or lagoons featuring a paddle wheel that circulates wastewater. HRAPs
can be an economical and sustainable method for treating wastewater, as microalgae efficiently
absorb nutrients such as phosphorus and nitrogen, as well as help remove organic and inorganic
contaminants [29]. Outdoor HRAPs are heavily influenced by various uncontrollable environmental
factors, such as seasonal changes and weather conditions [30]. An inconvenience of this cultivation
method is that open pond cultivation of mutant microalgal strains poses environmental and
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industrial risks. The release of mutants could damage local biodiversity and increase the probability
of algal blooms.
3.2. Close Photoreactors
Closed photobioreactors (PBR) are enclosed systems utilized for the cultivation of microalgae
and other phototrophic microorganisms. They provide excellent control over culture conditions with
minimal risk of contamination. Different types of PBRs include flat panel, tubular, and stirred tank
designs [31]. Many studies have employed PBRs with Chlamydomonas, primarily for basic research
purposes. Next, we will focus on studies that have yielded practical or biotechnologically promising
applications. The maximum biomass productivities were investigated in C. reinhardtii using two
different PBRs: a torus-plane and a cylindrical reactor. The research highlighted that optimizing the
design of Chlamydomonas PBR involves managing three key parameters: the specific illuminated area,
the illuminated working volume fraction, and the mean value of the incident hemispherical photon
flux density [32]. Additionally, optimization of a pilot-scale photobioreactor (120 L) for
Chlamydomonas found that the gas diffuser design significantly impacted biomass production [33]. A
110 L PBR made of polypropylene and transparent Plexiglas, consisting of 64 tubes arranged in an
8x8 square pitch cell connected by U-bends, with a total length of 133 meters, has been specially
designed for C. reinhardtii [34]. Recent studies have investigated the potential of utilizing the
phototactic response of C. reinhardtii to reduce the economic cost of mixing in PBR. By exploiting its
phototactic mechanism, C. reinhardtii can be stimulated to swim in opposite directions, thus
providing mixing and ensuring access to nutrients without mechanical agitation, thereby enhancing
PBR energy efficiency [35]. A multi-scale PBR, Antares I, with incident light modulations ranging
from 10 to 300 µmol photons m−2 s−1, has been tested with C. reinhardtii and has shown improvement
in biomass production compared to traditional PBRs. With Antares I the estimated doubling time for
Chlamydomonas culture is half of that reported for culture using similar media and light conditions
[36]. These findings demonstrate the diverse applications and research efforts related to the
optimization of PBR for Chlamydomonas cultivation.
3.3. Biofilm Reactors
The cultivation of microalgae in biofilm reactors involves immobilizing the microalgae on a
surface that acts as a support, forming a continuous layer. This method offers advantages such as
higher concentration per unit volume of medium, reduction or absence of cells in the effluent, and
ease of harvesting [37]. The extraction and dewatering of algae cells from biofilms are simplified by
the ease of separating attached cells from their growth medium. In the context of technological
applications, regulating the adhesion properties of Chlamydomonas could significantly enhance the
efficiency of biofilm reactors by controlling surface colonization and biofilm formation. So far, the
basic principles governing the colonization of surfaces by motile, photosynthetic microorganisms
remain largely unexplored. Research has shown that the surface adhesion of C. reinhardtii is flagella-
mediated and largely substrate-independent, enabling it to adhere to any type of surface [38].
However, it has been shown that the biofilm adhesion of C. reinhardtii is controlled by the type of
light, being activated in blue light and deactivated under red light [39]. Interestingly, Chlamydomonas
has the ability to secrete substances such as sulphated polysaccharides that act as antibiofilm agents
for certain bacteria, preventing these bacteria from attaching to the biofilm [40]. This property can be
highly beneficial in controlling the occurrence of bacterial contaminations.
3.4. Encapsulation
The encapsulation of microalgae is a process in which the microalgae are coated with a protective
layer to enhance their stability, protect them from adverse conditions, and facilitate their application.
This process offers various biotechnological advantages, such as protecting the formation of bioactive
compounds, promoting release control, improving solubility, and enhancing bioavailability [41].
Various materials, including alginate, carrageenan, chitosan, and polyvinyl, have been used for the
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immobilization of microalgae [42]. In the case of Chlamydomonas, alginate has been the most
successful and currently the most commonly used material for encapsulation. Some of its properties
include low cost, biocompatibility, transparency, permeability, and mechanical defense, which
reduces the risk of contamination [43]. The pore size of alginate beads in C. reinhardtii is critical, with
the highest efficiency for contaminant removal obtained in gel beads with a pore size of 3.5 mm [44].
Silica hydrogels have been utilized to entrapped C. reinhardtii cells, offering advantages over alginate,
such as greater stability against microbial attacks, and higher transparency [45]. One drawback of
alginate encapsulation is its high porosity, which can lead to the release of large molecules. However,
it has been observed with C. reinhardtii that the combination of alginate and silica to create hybrid
beads can provide superior properties that overcome these limitations [46]. Single-cell encapsulation
using metal-phenolic networks is an innovative technique aimed at protecting cells from stressors.
This method involves coating individual cells with metal-phenolic networks to create a mechanical
barrier. It was first employed with C. reinhardtii, finding that this type of encapsulation served as a
mechanical barrier, delaying the proliferation of the coated cells and effectively promoting
flocculation [47].
4. Chlamydomonas Phycoremediation
Microalgae, particularly Chlamydomonas, exhibit a remarkable capacity and diversity in
bioremediating various molecules. Next, we will present the main mechanisms for bioremediation.
Biosorption is a passive mechanism whereby microalgae serve as a biological sorbent to capture and
accumulate pollutants. Microalgae utilize their cell wall and various chemical groups to attract and
retain contaminants [48]. Microalgae can remove pollutants through bioaccumulation. The main
differences between biosorption and bioaccumulation processes lie in their mechanisms. Biosorption
is a passive process where microorganisms utilize their cellular structure to capture pollutants on the
binding sites of the cell wall. On the other hand, bioaccumulation is an active process that involves
the accumulation of pollutants in the biomass of microalgae, either by accumulation or uptake into
intracellular spaces [49]. Bioaccumulation requires cellular growth and is typically slower than
biosorption. Biotransformation involves the breakdown of pollutants, either inside or outside the
cells, facilitated by enzymes [50]. While there aren't significant concerns with biosorption and
bioaccumulation, biotransformation presents more challenges due to the possibility of its products
being potentially more toxic than the original compounds.
Some studies have cultivated Chlamydomonas in PBRs for the decontamination of wastewater. In
this regard, Chlamydomonas debaryana using dairy wastewater reduced nitrogen, phosphorus, organic
carbon, and chemical oxygen demand by more than 85% [51]. C. debaryana and C. reinhardtii were able
to effectively treat swine wastewater [52]. With C. reinhardtii, 55.8 mg of nitrogen and 17.4 mg of
phosphorus per liter per day were effectively removed from industrial wastewater [53]. Wastewater
collected from a paper industry was treated using C. reinhardtii, resulting in significant reductions of
nitrate (86%), phosphate (88%), and chemical oxygen demand (93%) [54]. Using C. mexicana, a high
removal efficiency of nitrogen (62%), phosphorus (28%), and inorganic carbon (29%) was achieved in
piggery wastewater [55].
Numerous studies have reported the use of HRAP in wastewater treatment, primarily focusing
on genera such as Scenedesmus and Chlorella [56]. However, very few records exist of applying HRAP
with Chlamydomonas. In a pilot-scale HRAP experimental wastewater treatment, Chlamydomonas sp.
was found to be one of the dominant genera. The study reported a reduction of the biochemical
oxygen demand by 90%, chemical oxygen demand by 65%, total nitrogen by 46% and total
phosphorus by 20% [57]. A study on the bioremediation of piggery wastewater using HRAP revealed
that Chlamydomonas sp. was the dominant species, with average chemical oxygen demand and total
nitrogen removal efficiencies of 76% and 88%, respectively [58]. In another study employing HRAP
with Chlamydomonas sp. for treating municipal wastewater, average reductions of volatile suspended
solids, total nitrogen, and biochemical oxygen demand were 63%, 76%, and 98%, respectively [59].
Chlamydomonas sp. JSC4 has been successfully employed in a biofilm reactor for the removal of
phosphorus, nitrogen, and copper from swine wastewater [60]. In a biofilm reactor, Chlamydomonas
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pulvinata TCF-48 g has demonstrated significant polyphosphate accumulation and a high phosphorus
removal rate of 70%, making it valuable for phosphate recovery applications [61]. The encapsulation
of C. reinhardtii in alginate beads has been successfully carried out to remove various types of
contaminants such as phosphorus, nitrogen, lead, mercury and cadmium [62] or even phenol [63].
C. reinhardtii has shown a significant capability for biosorption, effectively removing copper,
boron, and manganese [64], arsenic [65], nickel [66], zinc, cadmium [67] and uranium [68]. In C.
reinhardtii gene manipulation has been conducted to enhance the expression of the metal tolerance
proteins metallothioneins [69], resulting in increased tolerance to cadmium [70], chromium [71],
copper [72], mercury [73], and lead [74]. Biosorption in C. reinhardtii as a defense mechanism against
silver nanoparticles involves an increase in phytochelatin and exopolysaccharides content, along
with a decrease in glutathione levels [75]. C. reinhardtii has been shown to bioaccumulate several
compounds as Prometryne (herbicide) [76], o-nitrophenol [77], and C. mexicana carbamazepine
(antiepileptic agents) [78].
Some of the pollutants removed by biotransformation by C. reinhardtii include
organophosphorus pesticide such as trichlorfon [79], polycyclic aromatic hydrocarbons such as
benz(a)anthracene [80], polystyrene [81] and microplastics as bisphenol A [82]. The pharmaceuticals
products that can be biotransformed by microalgae have been reviewed in [83]. Among these,
Chlamydomonas has demonstrated high efficiency with the following compounds: Chlamydomonas sp.
with 7-amino-cephalosporanic acid [84], C. mexicana with enrofloxacin [85], and C. reinhardtii with
carbamazepine, ciprofloxacin, erythromycin, estrone, norfloxacin, ofloxacin, paracetamol,
progesterone, roxithromycin, salicylic acid, sulfadiazine, sulfadimethoxine, sulfametoxydiazine,
sulfamethazine, triclocarban, triclosan and trimethoprim [86], sulfadiazine [87] and ibuprofen [88].
C. reinhardtii has been found to biotransform antibiotics like azithromycin, erythromycin, and
sulphapyridine [89]. C. reinhardtii was shown to be able to biotransform the hormones β-estradiol and
17α-ethinylestradiol [90] as well as the non-steroidal anti-inflammatory drug diclofenac [91].
Chlamydomonas can metabolize xenobiotics through a wide range of enzymatic processes,
including CYP450 oxidation reactions, hydrolysis, glutamate conjugation, and methylation [92].
Chlamydomonas moewusii excretes laccases capable of breaking down and detoxifying phenolic
pollutants [93]. The toxicity responses of different pollutants, such as benzophenone-3, bisphenol A,
oxytetracycline, and atrazine, in C. reinhardtii showed a similar pattern: an increase in chlorophyll
autofluorescence and a decrease in growth rate and vitality [94]. The biotransformation of five
bisphenol derivatives (AF, B, F, S, and Z) by C. mexicana shows that all the biotransformed products
were less toxic than the parent compounds [95]. Chlamydomonas has also been used in efforts to
degrade commonly used plastic components such as Polyethylene terephthalate (PET). In Ideonella
sakaiensis, a novel plastic degradation enzyme called PETase has been identified [96]. The I. sakaiensis
PETase has been expressed through genetic recombination in the C. reinhardtii nucleus and
chloroplast genomes, showing a significant ability to break down PET [97]. Under specific adverse
conditions such as NaCl stress, EDTA exposure or acidic pH, C. reinhardtii can form multicellular
aggregates called palmelloids. These are small clonal structures that result from cells failing to
separate after division [98]. The defense mechanisms of C. reinhardtii under perchlorate stress were
investigated, revealing palmelloid formation when exposed to 100 and 200 mM perchlorate [99].
These researchers highlight the metabolic versatility of Chlamydomonas in dealing with xenobiotic
compounds, demonstrating its ability to transform and process a variety of chemicals through
different mechanisms.
Microalgae have been actively employed in initiatives focused on reducing CO2 emissions due
to their ability to absorb CO2 via photosynthesis. C. reinhardtii exhibits a superior ability to fix CO2
compared to other photosynthetic organisms [100]. Bio-fixation refers to the process by which certain
organisms, such as microalgae, utilize CO2 from the air or other sources like flue gas streams to create
biomass. The production of 1 gram of microalgae biomass leads to the sequestration of 1.8 grams of
CO2 [101]. In Chlamydomonas the expression of a single H+-pump increase its tolerance to high
concentrations of CO2, such as those found in industrial flue gas [102]. These findings illustrate the
potential of C. reinhardtii to mitigate CO2 emissions from industrial sources.
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5. Chlamydomonas Bioproduct Generation
5.1. Biomass
One of the main products derived from the cultivation of microalgae is their biomass, as it is
used as raw material for obtaining other derived bioproducts. It would be highly beneficial
economically to use the biomass resulting from the bioremediation process for bioproduct
purification. However, the utilization of biomass derived from wastewater treatment encounters
several inherent challenges. These challenges include the scalability of biomass production, the
presence of xenobiotics and heavy metals, as well as the contamination with bacteria, fungi, and
viruses, all of which limit their extensive application [103]. Although numerous efforts are being
made to address this issue, the production of the main bioproducts obtained from microalgae still
does not use wastewater as a cultivation source. Next, we will present studies that utilize
Chlamydomonas to obtain certain bioproducts, some of which use biomass derived from wastewater
remediation.
The composition of biomass is influenced by the strains of microalgae and the culture conditions
[104]. One straightforward method to increase biomass productivity involves altering the culture
medium conditions or adjusting the supply of certain macroelements. For example, in microalgae,
some researchers have evaluated the effect of various carbon sources [105], pH variations [106], the
photoperiods [107], as well as trace elements composition for biomass production [108].
Consequently, various approaches have been explored to optimize microalgae biomass enriched in
specific biomolecules (Figure 2). The highest biomass concentration of Chlamydomonas obtained so far
has been heterotrophically with acetate, reaching 23 g/l [109], far behind compared to other green
algae that are able to consume glucose as a substrate, like Chlorella sp. and Scenedesmus sp., for which
biomass reached 271 g/L and 286 g/L respectively [110].
Figure 2. The process of Chlamydomonas bioproduct generation. Chlamydomonas cells can be cultivated
in various conditions. Cultivation optimization is a crucial factor and involves processes such as strain
selection, nutrient availability, temperature control, light intensity, salinity, and pH levels, which
impact the algae’s growth and productivity. After cultivation, the collected biomass and medium can
then be processed as indicated to obtain the specified bioproducts.
5.2. Biochar
Biochar is a carbonaceous material produced through the pyrolysis of biomass (Figure 2), which
can be obtained from microalgae, agricultural residues, wood, or organic waste [111]. Biochar is
characterized by its high porosity and specific surface area, making it useful for improving soil
quality and carbon sequestration. It is used in agriculture as a soil amendment to enhance soil
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structure, retain nutrients and water, and promote beneficial microbial activity. Additionally, biochar
is considered a strategy for mitigating climate change, as burying it in the soil can store carbon stably
for long periods [112]. C. reinhardtii biomass has been successfully used to prepare biochar [113]. The
highest biochar yield was 93.9%, achieved by dry torrefaction at 200°C of Chlamydomonas sp. JSC4
[114]. Biochar prepared from Chlamydomonas sp. has been shown to have a high capacity for removing
contaminants [115].
5.3. Biofertilizers
Microalgae are used as a biofertilizers and biostimulants by promoting crop growth and
increasing soil nutrient contents, thereby reducing the usage of chemical fertilizers [116]. In contrast,
Chlamydomonas species have received little attention and are not fully utilized in agriculture, despite
being among the most abundant microalgae species in natural soil ecosystems. In this regard, a study
on the effects of Chlamydomonas applanata M9V as a biofertilizer on wheat found that it performed
even better than a certain amount of chemical fertilizer [117]. Acid-hydrolyzed dry biomass of C.
reinhardtii improved the phosphorus, nitrogen, and carotenoid contents of Solanum lycopersicum [118].
The application of live Chlamydomonas cells significantly increased leaf size, shoot length, fresh
weight, number of flowers, and pigment content of Medicago truncatula [119]. Lyophilized powders
derived from C. reinhardtii have been found to positively affect the growth of maize plants by
producing bioactive compounds that act as biostimulants, enhancing plant growth, crop
performance, yields, and quality [120]. Biomass extracts of Chlamydomonas sp. exhibited auxin-like
activity that increased the number of roots in cucumber [121]. Chlamydomonas sajao can improve soil
physical properties, such as aggregation and stability, thereby contributing to enhanced soil structure
and nutrient retention [122]. These results suggest that Chlamydomonas can be an effective alternative
to chemical fertilizers for promoting crop growth and yield.
5.4. Bioplastic
Bioplastics are biodegradable materials derived from renewable biomass sources, offering a
sustainable alternative to traditional plastics [123]. Various molecules can be used as building blocks
for bioplastics, including polyhydroxybutyrate (PHB), starch, TAG, lactic acid, or polybutylene
succinate. PHB can be naturally synthesized by certain bacteria, such as Azotobacter or Pseudomonas.
PHB production involves three key enzymes: β-ketothiolase, acetoacetyl-CoA reductase, and PHB
synthase, encoded by phbA, phbB, and phbC, respectively. Research has focused on engineering
Chlamydomonas strains to enhance PHB de novo biosynthesis, as Chlamydomonas naturally cannot
synthesize PHB. With this aim, the phbB and phbC genes from Ralstonia eutropha have been inserted
into the C. reinhardtii genome, leading to the observation of PHB granules in the cytoplasm [124].
While cytosolic accumulation of PHB in Chlamydomonas often results in impaired cell growth and low
yield, peroxisomes have emerged as a promising alternative. A complete PHB biosynthesis pathway
has been successfully reconstructed by expressing the three PHB synthesis genes and targeting the
proteins to the peroxisomes. Within the peroxisomes of these strains, PHB reached 21.6 mg/g, which
represents a 3600-fold increase over cytosolic PHB production [125]. Another strategy is to use TAG
as the building block for bioplastic. TAG synthesized by C. reinhardtii has been directly crosslinked
with glycerol or ammonium persulfate and molded into plastic beads that are capable of
withstanding compressive stress up to 1.7 megapascals [126]. Cell-plastics are a type of bioplastic that
directly utilizes raw cells and the hydrolyzed cell broth. Unlike conventional bioplastics, cell-plastics
do not require exhaustive processes for extracting and refining the biomolecules that serve as the
building blocks. Recently, Chlamydomonas cells have arisen as the constituent blocks of this new type
of bioplastic, as their cell size and protein-rich, cellulose-free cell wall were demonstrated to be ideal
components for its fabrication [127].
5.5. Biofuels
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Biofuels are fuels derived from renewable biological sources such as plants or plant-derived
materials. First-generation biofuels are produced from food crops. Second-generation biofuels are
derived from non-food sources such as waste, and third-generation biofuels are produced from
sources that do not compete with arable land, such as microalgae [128]. Microalgae have regained
attention as alternative resources for environmentally friendly production of biofuels, including
biodiesel, bioethanol, biogas, and biohydrogen. These biofuels can be produced through
thermochemical and biochemical conversions, photosynthesis-mediated microbial fuel production,
and transesterification [129].
5.5.1. Biodiesel
Triacylglycerols (TAG) are crucial lipids in microalgae for biofuel production. Oleaginous
microalgae, rich in TAG, can be converted into biodiesel through transesterification, a process that
transforms TAG into fatty acid methyl esters, the key components of biodiesel [130]. Utilizing
Chlamydomonas sp. JSC4, a direct transesterification process was employed, resulting in nearly 100%
biodiesel production in a single step [131]. Given that biodiesel production is closely linked to the
quantity of lipids and TAGs, various strategies have been explored to enhance their production in
Chlamydomonas. Some studies have focused on elucidating the functions of key genes involved in
lipid and TAG production. The down-regulation of the phosphoenolpyruvate carboxylase gene in C.
reinhardtii resulted in a 74.4% increase in lipid content [132]. The overexpression of acetyl-CoA
synthetase resulted in a 2.4-fold increase in the accumulation of TAG [133]. In C. reinhardtii, the
mutation of ACX2, which encodes a member of the acyl-CoA oxidase responsible for the first step of
peroxisomal fatty acid beta-oxidation, resulted in an accumulation of 20% more lipid [134]. A mutant
of C. reinhardtii deficient in phospholipase showed an increase in TAG content of up to 190% [135].
The overexpression of the ferredoxin gene PETF in C. reinhardtii resulted in higher lipid content [136].
The Target of Rapamycin (TOR) plays a crucial role in regulating cell growth. It has been shown that
mutants of C. reinhardtii lacking TOR experience an increase in TAG production [137]. The strategy
of heterologously overexpressing genes in Chlamydomonas has been successful in increasing TAG
content. In this sense, the heterologous expression of the Dunaliella tertiolecta fatty acyl-ACP
thioesterase in C. reinhardtii leads to increased lipid production [138]. By expressing the diacylglycerol
acyltransferase from Saccharomyces cerevisiae into C. reinhardtii, the fatty acids and TAG content
increased by 22% and 32%, respectively [139]. The heterologous expression of Lobosphaera incisa
glycerol-3-phosphate acyltransferase in C. reinhardtii enhances TAG production [140]. The synthesis
of starch and lipids competes for carbon skeletons; thus, inhibiting starch synthesis is another strategy
followed to increase TAG production. In this sense, silencing ADP-glucose pyrophosphorylase in C.
reinhardtii resulted in a tenfold increase in TAG content [141]. Genetically modifying Chlamydomonas
sp. JSC4 in the gene that encodes the starch debranching enzyme promotes carbohydrate degradation
and redirects carbon resources into lipids, resulting in a 1.46-fold increase in lipid [142].
A commonly employed approach to accumulate TAG in Chlamydomonas is to induce stress
conditions, particularly nutrient limitation or starvation [143]. C. reinhardtii exhibits a notable increase
in TAG accumulation under low nitrogen concentration [144]. Under nitrogen deprivation, C.
reinhardtii starch mutants exhibit almost a 10-fold increase in TAG [145]. Under nitrogen limitations,
increasing the expression of S-adenosylmethionine synthetase in C. reinhardtii enhances cell viability
and TAG production [146]. Phosphorus stress also triggers TAG production in Chlamydomonas [147].
Additionally, a higher TAG content is generated under conditions of low sulfur concentration [148].
The lipid content in C. mexicana was observed to rise as the concentration of NaCl was increased to
25 mM [149]. The lipid content of the C. reinhardtii starchless mutant BAF-J5 increased by 76%
following a temperature shift to 32°C [150].
Increasing TAG levels by inducing stress conditions often comes at the expense of inhibited
microalgal growth. Under these conditions, there is an inverse relationship between TAG yield and
microalgal growth. To mitigate this, it has been reported that overexpressing the transcription factor
MYB1 in C. reinhardtii, which mediates lipid accumulation, results in nearly 60% more TAG without
negatively impacting cell growth [151]. In another strategy, a cultivation approach involving two
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stages has been proposed, wherein C. reinhardtii experiences nutrient stress only after an initial period
of optimal growth, allowing for high TAG accumulation [152]. The development of effective methods
for cultivating Chlamydomonas is essential in biodiesel production. In this regard, in C. reinhardtii a
multi-parametric kinetic model developed using computational tools has been proven, resulting in
significant increases in lipids (74%) [153].
5.5.2. Bioethanol
Bioethanol is a biofuel that can be obtained through the fermentation of various types of biomass
containing high amounts of sugars. For bioethanol production, the high carbohydrate content present
in both the cellulose and hemicellulose cell walls, as well as the starch-based cytoplasm, is broken
down into monomeric sugars during enzymatic hydrolysis prior to fermentation. However, the cell
wall of Chlamydomonas is not made of cellulose like in plants, but of five dense glycoprotein-rich
layers [154]. Therefore, efforts have been focused on utilizing starch-rich Chlamydomonas for the
production of bioethanol. The biomass of C. reinhardtii UTEX 90 was converted into glucose through
two hydrolytic steps using α-amylase and amyloglucosidase, with nearly all the starch successfully
transformed into glucose without damaging the cell wall, reducing the costs of bioethanol
purification [155]. Pretreating C. reinhardtii UTEX 90 biomass with sulfuric acid (1-5%) at
temperatures ranging from 100 to 120°C significantly increases the glucose release for the production
of bioethanol [156]. The psychrophilic alga Chlamydomonas sp. KNM0029C was studied for its
potential to produce bioethanol, showing promising results (Kim et al., 2020). The supraoptimal
temperature treatment method, which involved cultivating C. reinhardtii at 39°C despite its optimal
temperature being 25°C, was successfully applied and resulted in nearly a threefold enhancement of
starch content [157]. In C. reinhardtii, optimizing the pH, temperature, initial concentrations of acetate
and ammonium, along with the use of depigmented and defatted biomass, led to a bioethanol yield
ranging from 90% to 94% (Banerjee et al., 2021). The hormones have also been described to have a
very important role in starch accumulation; in this sense, in Chlamydomonas 100 µM of Indole-3-acetic
acid produces an accumulation of up to nine times more starch [158]. Chlamydomonas sp. QWY37 has
been effectively utilized for bioethanol production from swine wastewater, achieving a maximum
bioethanol yield of 61 g/L [159].
5.5.3. Biogas
Biogas is a renewable energy source primarily composed of CH4, derived from the microbial
anaerobic digestion of biomass obtained from various sources (Figure 2). The production of biogas
involves multiple stages, including hydrolysis, acidogenesis, acetogenesis, and methanogenesis,
which are facilitated by a microbial consortium that plays a crucial role in influencing both the
composition and yield of the biogas [160]. This process eliminates the need to extract specific
macromolecules, such as lipids, proteins, or carbohydrates, and can be carried out using wet biomass
[161]. The fermentation of C. reinhardtii biomass produces approximately 587 ml of biogas per gram
of volatile solids [162]. However, microalgae biomass is not ideal for biogas generation due to its high
protein content, which results in an unfavorable low carbon-to-nitrogen ratio. This imbalance arises
because the ammonia released during protein degradation inhibits the methanogenesis process [163].
C. reinhardtii biomass has been studied for its potential in overcoming this limitation. In this regard,
the anaerobic digestion of C. reinhardtii biomass obtained in low nitrogen media has shown
remarkable efficiency in biogas production due to its high carbon-to-nitrogen ratio [164].
The high resistance of microalgae biomass to microbial decomposition due to their rigid cell
walls is a significant challenge in biogas production. However, since the main components of the C.
reinhardtii cell wall are glycoproteins rather than cellulose, C. reinhardtii has been shown to produce
larger quantities of biogas compared to species with more complex cell walls (such as Chlorella sp.
and Scenedesmus sp.) [165]. The findings revealed that the C. reinhardtii cell wall was not an obstacle
but instead became advantageous by enabling the gradual degradation of intracellular content [166].
One way to valorize the microalgal biomass produced during wastewater treatment is to utilize it as
a source for biogas production, thereby reducing the economic costs of treatment [167]. In this regard,
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Chlamydomonas sp. Ck has demonstrated high efficiency in decontaminating piggery wastewater
while simultaneously producing a high biogas yield [168]. For all the reasons mentioned, anaerobic
digestion of C. reinhardtii biomass can be considered a cost-effective alternative for biogas production
compared to other methods.
5.5.4. Hydrogen
The production of the preceding bioproducts shares the common step of first obtaining biomass,
and then extracting these compounds from it. Next, we will present some products that
Chlamydomonas releases into the culture medium and therefore can be purified without needing to be
extracted from the biomass, thereby reducing the economic cost of their production (Figure 2). A
prominent example of this is hydrogen, that has emerged as one of the most promising energy
carriers for future energy demands. Hydrogen presents the opportunity to cultivate living organisms
such as bacteria, cyanobacteria, and microalgae capable of releasing H2 into the media [169].
Hydrogen is generated through enzymes known as hydrogenases [170]. Chlamydomonas has two
hydrogenases that have been extensively studied with the aim of increasing their production
efficiency [171]. The hydrogenases catalyze the reduction of protons into H2 either using energy from
light (biophotolysis) or by oxidizing organic compounds such as starch (dark fermentation). One of
the primary biotechnological challenges of using Chlamydomonas as a factory to produce H2 is the
rapid inactivation of its hydrogenases by oxygen, particularly considering that oxygen is generated
during photosynthesis. Therefore, the initial evidence indicating that Chlamydomonas was capable of
producing H2 was observed with Chlamydomonas moewusii under anaerobic condition [172], and
subsequently with C. reinhardtii, also anaerobically [173]. The first successful strategy demonstrating
significant and consistent H2 production under aerobic conditions involved using sulfur-starved C.
reinhardtii [174]. The reason for this is that the absence of sulfur blocks protein synthesis, thereby
halting photosynthesis and oxygen production. Alternative strategies for H2 production under non-
stress conditions are also possible, particularly in media containing acetate, which is compatible with
Chlamydomonas growth [175,176]. However, the rates of H2 production under non-stress conditions
are lower compared to those under stressful condition [177].
In Chlamydomonas, numerous genetically engineered strains have been developed to enhance H2
production. One of the most successful approaches has been to improve the intrinsic oxygen tolerance
of hydrogenase through mutagenesis [178]. A production of 1200 mL of H2 per liter has been reported
after 6 days using the Photosystem I (PSI) cyclic electron transport mutant pgr5, which is defective in
thylakoid proton gradient regulation [179]. Another strategy is diverting electron flow to the
hydrogenase [180], and degrading or inhibiting the function of Photosystem II (PSII) to prevent
oxygen production [181]. However, strategies that do not degrade PSII appear to be advantageous,
as the long-term loss of PSII inhibits cell growth. In this sense, a PSI-hydrogenase chimera was created
by inserting the HydA sequence into the PsaC (stromal subunit of PSI). This redirects photosynthetic
electron flow towards proton reduction [182]. A disadvantage in the use of Chlamydomonas is that the
hydrogen production rate is influenced by the size of microalgae cells. The hydrogen production rate
of Chlorella is higher than that of Chlamydomonas due to its relatively smaller size [183].
5.6. High-Value Bioproducts
The term "high-value bioproducts" refers to a wide range of products derived from various
sources, which economically have a higher value compared to low- to medium-value products. C.
reinhardtii is a promising organism for the production of high-value bio-products [184]. Glycolate, a
high-value cosmetic ingredient can be overproduced in Chlamydomonas. When Chlamydomonas is in
an environment with low CO2 (0.04%), Rubisco oxygenates ribulose-1,5-bisphosphate instead of
carboxylating it, consequently producing glycolate. In Chlamydomonas, glycolate is toxic, prompting
an active system to excrete it. To facilitate the recovery of potentially lost carbon, the genes for
photorespiratory metabolism are induced. Photorespiration detoxifies and recycles glycolate,
generating glycerate and releasing CO2. In Chlamydomonas, glycolate dehydrogenase (GDH) is
involved in photorespiration by oxidizing glycolate to glyoxylate. It has been observed that
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Chlamydomonas GDH mutants over-accumulate glycolate in the media [185]. Chlamydomonas has a
CO2-concentrating mechanism (CCM) to prevent the rubisco oxygenation reaction and, consequently,
glycolate excretion [186]. CIA5 is the primary transcription factor that induces the CCM, and its
mutation has been shown to increase the amount of excreted glycolate [187]. By incorporating 6-
Ethoxy-2-benzothiazolesulfonamide (EZA), a CCM inhibitor, glycolate production can be maximized
without compromising cell viability. Under these conditions, glycolate accumulates in the medium,
reaching a concentration of up to 41 mM [188]. In photorespiration, hydroxypyruvate is converted to
glycerate by hydroxypyruvate reductase (HPR). In C. reinhardtii, the mutation of hpr1 results in
increased excretion of glycolate into the medium [189].
Bioisoprenoids are natural compounds synthesized by plants, animals, and microorganisms
through the isoprenoid biosynthetic pathway. These compounds are structurally and functionally
diverse, with a wide range of applications, including their use as perfumes, cosmetics, pigments,
medicines, and chemical signals. Bioisoprene production has gained attention due to its sustainability
and efficiency compared to petrochemical sources [190]. It has been demonstrated that C. reinhardtii
can be genetically modified to produce significant amounts of bioisoprene by overexpressing four
different plant isoprene synthase genes (IspS), with the strain expressing the Ipomoea batatas IspS gene
showing the highest isoprene levels [191].
Hydroxyalkanoyloxyalkanoates (HAA) are a type of lipidic surfactants that can be produced by
certain bacteria that show great potential for a wide range of applications. They are synthesized by
the condensation of hydroxyalkanoic acids, which are produced by the metabolism of fatty acids. The
chloroplast genome of C. reinhardtii was engineered by inserting the gene encoding the
acyltransferase of P. aeruginosa, a key enzyme in HAA synthesis, resulting in high concentrations of
HAA not only in the intracellular fraction but also in the extracellular [192].
There is strong interest in developing bio-based hydrocarbons and their unsaturated analogs,
the alkenes, as potential substitutes for hydrocarbons derived from petroleum. The alkene 7-
heptadecene has high demand for various biotechnological processes. While the biological function
of alkenes in microalgae remains completely unknown, it has been shown that in C. reinhardtii, the
enzyme fatty acid photodecarboxylase is responsible for synthesizing 7-heptadecene [193]. This
discovery opens the possibility of overproducing this alkene in C. reinhardtii. ε-Polylysine is a
biodegradable polymer composed of 25-30 lysine monomers that has a variety of applications,
including antimicrobial activity and anticancer agent [194]. It has been reported that ε-polylysine is
produced from Chlamydomonas sp. supplemented with lysine, aspartate, and tricarboxylic acids,
achieving a maximum production of 2.24 g/L [195].
Bio-polyamides, also known as nylons, are sustainable polymers derived from renewable
resource. Bio-polyamides have excellent material properties, leading to a high demand for polyamide
plastics with diverse applications across various industries [196]. Cadaverine and putrescine are
polyamines commonly used as precursors and building block for the synthesis of bio-polyamides. By
the heterologous expression of two E. coli lysine decarboxylases in C. reinhardtii, it was possible to
significantly enhance the synthesis of cadaverine [197]. The mutation of essential genes in the C.
reinhardtii polyamine biosynthesis pathway identified ornithine decarboxylase 1 (ODC1) as a crucial
regulator that controls the accumulation of putrescine. Subsequently, the authors overexpressed
different ODCs, resulting in a significant increase in cellular putrescine levels, reaching a maximum
yield of 200 mg/L [198]. This achievement marks the first instance of microalgal bio-production of
putrescine.
C. reinhardtii, along with Chlorella vulgaris, Dunaliella bardawil, Arthrospira platensis,
Auxenochlorella protothecoides, and Euglena gracilis are among the very few microalgae recognized
by the Food and Drug Administration as Generally Recognized as Safe (GRAS) organisms (GRAS
Notice No. 773). This acknowledgment allows their use as a nutritional component in food,
presenting new opportunities for the utilization of C. reinhardtii. Clinical studies on the human
consumption of C. reinhardtii whole cells have demonstrated positive effects on gastrointestinal
health and microbiota, showing that the intake of C. reinhardtii cells promotes microbiota eubiosis,
reducing imbalances and improving the overall health of the intestine [199]. The development of
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alternative plant-based products to substitute meat has led to the exploration of heme-containing
proteins for their ability to provide a meat-like color and flavor. One such compound that can provide
these qualities is protoporphyrin IX (PPIX) a crucial intermediate in the heme biosynthetic pathway.
In this regard, engineered C. reinhardtii strains have been shown to overexpress PPIX [200].
Antioxidants are widely recognized for their beneficial impact on health and their crucial role in
protecting cells from the harmful effects of free radicals. Chlamydomonas agloeformis has garnered
attention due to its exceptionally high antioxidant capacities that surpass those of higher plants [201].
Carotenoids are a diverse group of lipid-soluble pigments produced by plants and microorganisms,
known for their benefits as vitamin precursors and antioxidants. Astaxanthin, a ketocarotenoid, is
recognized as one of the most powerful natural antioxidants among carotenoids [202]. Astaxanthin
is currently primarily produced industrially from the microalgae Haematococcus pluvialis, with the
crucial enzyme involved in its biosynthesis being β-carotene ketolase (BKT)[203]. The synthetic
redesign and overexpression of C. reinhardtii BKT has been shown to achieve Astaxanthin
productivities of up to 4.3 mg/L/day, which is comparable to the results obtained with H. pluvialis
[204]. This production does not impair the growth or biomass productivity of C. reinhardtii, presenting
a promising alternative to natural astaxanthin-producing algal strains. Furthermore, the
accumulation of astaxanthin has led to enhanced high light tolerance and increased biomass
productivity [205]. Blocking the expression of ATG1 and ATG8, genes involved in autophagy in C.
reinhardtii, leads to a 2.3 times increase in carotenoid biosynthesis, indicating that autophagy does
play a role in regulating carotenoid levels [206].
Chlamydomonas has been shown to be able to synthesize vitamins C, A, E, B1, B7, B9, and
ergosterol, the precursor of vitamin D2 [207]. However, for most of these vitamins, the mechanisms
regulating their synthesis to achieve overproduction have not been studied in detail. In C. reinhardtii,
oxidative stress leads to a substantial increase in vitamin C levels [208]. Omega-3 fatty acids play
critical roles as nutrients and are extensively utilized in medicine. A comparison of C. reinhardtii with
Chlorella and Spirulina revealed that C. reinhardtii contains superior amounts of omega-3 fatty acids,
both in quality and quantity [209]. Sulphated polysaccharides (SPs) are polymer chains containing
one or more monosaccharide units that have been modified with sulfate groups. C. reinhardtii is
capable of synthesizing SPs, which have been associated with several beneficial properties, including
potent antioxidant and anticancer effects [210], antineurodegenerative [211] and antibiotics [212].
More than 40 therapeutic proteins, such as antibodies, enzymes, viral proteins, hormones,
among others, have been successfully expressed in C. reinhardtii [213]. ICAM-1, a protein belonging
to the immunoglobulin superfamily, was targeted for secretion into the extracellular media and was
found to be fully active, suggesting that C. reinhardtii can produce mammalian proteins that are
correctly folded and functional. Additionally, it achieved a concentration of up to 46.6 mg/L, marking
the highest reported concentration of any recombinant protein in C. reinhardtii to date [110]. The
production of full-length spike protein, a crucial component for the infectivity of SARS-CoV-2, has
been successfully achieved in C. reinhardtii as a secreted protein [214]. This achievement is crucial as
it offers a simpler and more economical platform for producing recombinant spike proteins in
microalgae.
6. Conclusions and Outlook
Throughout this review, various studies conducted with Chlamydomonas on bioremediation and
the production of bioproducts have been presented. As observed, the studies in this regard are
diverse and cover different fields. We believe that a key area to develop in the future, due to its
significant economic and environmental impact, would be to combine these two aspects
simultaneously. In other words, the biomass obtained from bioremediation should be used for the
production of a specific bioproduct of interest. As indicated in this review, some attempts have been
made in this direction, and although its development has significant challenges, we believe this
would be a very promising strategy to pursue in the future.
Author Contributions: AL original idea, conceptualization, and preparation of the first draft; AL, C B-P and MJ
T-P wrote the paper. All authors have read and agreed to the published version of the manuscript.
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Funding: This work was funded by Gobierno de España, Ministerio de Ciencia e Innovacion (Grant PID2020-
118398GB-I00), Junta de Andalucía (Grant ProyExcel_00483), the “Plan Propio” from University of Cordoba, and
a grant awarded by the Torres-Gutierrez foundation.
Data Availability Statement: All data required to evaluate the conclusions of this paper are included in the main
text.
Acknowledgments: This paper is dedicated to Emilio Fernandez Reyes, who has recently retired after almost 40
years of studying Chlamydomonas reinhardtii as a reference organism. He was the driving force that promoted
our research on Chlamydomonas, the pillar that allowed its advancement, and our great teacher whom we will
never be able to repay for all the learnings received. We also thank Maribel Macias for her constant technical
support.
Conflicts of Interest: The authors declare no conflict of interest.
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