**2. Taxonomy and biodiversity**

The order Cyanidiales from the class Cyanidiophyceae are a group of asexual, unicellular organisms that diverged from ancestral red algae around 1.3 billion years ago [4, 5]. These unicellular red algae were classified into three genera, *Cyanidium*, *Cyanidioschyzon*, and *Galdieria*. *Cyanidioschyzon merolae* is clearly recognizable thanks to its characteristic size and shape, but the other two algae are morphologically very similar. Until 1981 *Cyanidium caldarium* was used as a synonym for *Galdieria sulphuraria*, however, this species can grow only autotrophically while *G. sulphuraria* is also able to grow heterotrophically [6, 7]. Based on morphological, ultrastructural and ecophysiological studies the class Cyanidiophyceae was therefore instituted, containing the family Cyanidiaceae, including *Cyanidium caldarium* and the family Galdieriaceae, including *Galdieria sulphuraria* [8].

Different Cyanidiophycean species, including *Galdieria*, are found all over the world; however, their distribution is discontinuous as they are restricted to hot springs and geothermal habitats. This is related to the discontinuity of geothermal environments. Two decades ago, little was known about their biodiversity, their population structures, and the phylogenetic relationships of Cyanidiales.

Research based on environmental PCR studies revealed an unexpected level of genetic diversity among Cyanidiales. It was demonstrated that the Cyanidiales comprise a species-rich branch of red algae [9]. The high divergence rates in the Cyanidiales could be possibly explained by an elevated mutation rate in these taxa, resulting potentially from DNA damage in their extreme environments. The analyses also reject the putative mesophilic origin of Cyanidiales and suggest ancestral thermo-acidotolerancy of this lineage [9].

Sequencing of the *rbcL* gene with high sequence divergence within the genus has contributed to the taxonomy of *Galdieria* [10–12]. A cladogram defining molecular relationships among these algae shows that *Cyanidium caldarium* and *Cyanidioschyzon merolae* form a sister group relationship with *Galdieria* [11]. The genus *Galdieria* is divided into two clades, one of which includes *G. sulphuraria* accessions from Naples (Italy), California, and Yellowstone and the other one includes *G. sulphuraria* accessions from Java (Indonesia) and Russian species [11].

Based on molecular phylogenetics, three well supported *Galdieria* species exist: *G. maxima* Sentsova, *G. sulphuraria* (Galdieri) Merola (including two Russian species *G. partita* Sentsova and *G. daedala* Sentsova, described based on morphological differences) and *G. phlegrea* Pinto, Ciniglia, Cascone et Pollio [9, 13, 14].

Consequently, the main lineages were identified: *G. phlegrea* [14] comprising strains thriving in acidic non-thermophilic Italian sites; *G. sulphuraria*, a group that is geographically dispersed worldwide, including *G. sulphuraria* strains as well as *G. partita* and *G. daedala*, isolated from acid-thermal springs in Russia [15]. A final lineage contains the cosmopolitan species *G. maxima* [15], which is clustered independently from the *Galdieria* clade (*G. sulphuraria* + *G. phlegrea*) and shows an unexplained sister group relationship with the morphologically distinct *Cyanidioschyzon merolae* [5, 9, 10, 12, 16].

**107**

mitochondria [11].

*The Red Microalga* Galdieria *as a Promising Organism for Applications in Biotechnology*

Generally, *Galdieria* (Cyanidiales) is well known from Italy [5, 9, 14, 17] Yellowstone National Park, USA [18, 19], New Zealand [20], Iceland [10], and

Phylogenetic analyses of the *rbc*L gene also showed that *Galdieria* from the coal mining site at Ostrava, Czech Republic, belongs to the cosmopolitan species *G. sulphuraria*, for now the only eukaryotic organism forming visible biomass on a burning coal-waste heap [10]. This was the first evidence of this species growing in central Europe, and isolates were closely related to the Italian strains, together forming the continental European lineage of *G. sulphuraria* [21]*.* Another, nonthermophilic strain of *Galdieria*, also found in the Czech Republic [22], referred to as CCALA 965 (Culture Collection of Autotrophic Organisms, Institute of Botany, CAS, Třeboň, Czech Republic) was found to belong to the species *G. phlegrea*, so far

Morphology of the unicellular taxa Cyanidiales is relatively simple. Thick-walled cells are of a spherical shape and usually contain one chloroplast, 1–3 mitochondria,

Representatives of the order Cyanidiales are unparalleled among phototrophic microorganisms (eukaryotes) in their ability to thrive in acidic (pH 0.5–3.5) and high temperature (38–56°C) geothermal environments. Soils, sediments, and endolithic habitats around hot springs, boiling mud pools, and steaming fumaroles are typical for these extremophiles, which are dominant in local microbial communities [5, 9, 10, 14, 17–20, 25–30]. They are the principal photosynthetic organisms found in hot acidic waters [31], where even photosynthetic prokaryotes, such as the

*G. sulphuraria* is a unicellular, spherical, spore-forming red alga. In addition to acidophilic and thermophilic properties, it has the ability to grow phototrophically, mixotrophically and moreover heterotrophically while utilizing sugars, alcohols or amino acids [7, 35, 36]. *G. sulphuraria* cells are morphologically indiscernible from cells of *Cyanidium caldarium*, however, are well recognizable thanks to their ready ability to grow heterotrophically in the dark [16, 18]. In its natural environment, *G. sulphuraria* has a yellow to green color; however, when heterotrophically grown in liquid medium, it looks like yellow-green to dark blue-green. The cell size of *G. sulphuraria* is larger than that of *C. caldarium*. It reproduces by endospore formation ranging from four to thirty-two. As originally described, *G. sulphuraria* has a single, cup-shaped, parietally localized chloroplast [8] and includes a vacuole and

Morphological similarities between *G. sulphuraria* and *G. phlegrea* are so high that methods for recognition of these species, their habitats, and growth require-

*G. phlegrea* (**Figure 1**) prefers a relatively low temperature (25–38°C) for growth and inhabits rather dry endolithic sites with high acidity (pH 0.5–1.5) [9, 14, 22]. The Latin word *phlegreus* means volcanic, what is consistent with the specific locality, (Campi Flegrei, Naples, Italy), where the alga was found [14]. The locality provides diverse environmental conditions in the form of hot springs, streams, mud, rock walls, with different pHs and temperature ranges, producing the different micro-

Strain DB01 of *G. phlegrea* from the Tinto River (Spain) has typical coccoid cells with thick smooth cell walls. Mature cells reach the average size of 6.4 μm. The cell possesses a blue-green chloroplast without pyrenoids. Typical for the alga is asexual

*DOI: http://dx.doi.org/10.5772/intechopen.89810*

recently from the Czech Republic [21, 22].

known only from Italy [13, 14].

**3. Morphology and extremophile properties**

cyanobacteria, are completely absent [32–34].

ments, together with molecular analyses, are used.

habitats occurring in that site. [14].

a nucleus, a vacuole, and energy reserve products [8, 12, 15, 23, 24].

*The Red Microalga* Galdieria *as a Promising Organism for Applications in Biotechnology DOI: http://dx.doi.org/10.5772/intechopen.89810*

Generally, *Galdieria* (Cyanidiales) is well known from Italy [5, 9, 14, 17] Yellowstone National Park, USA [18, 19], New Zealand [20], Iceland [10], and recently from the Czech Republic [21, 22].

Phylogenetic analyses of the *rbc*L gene also showed that *Galdieria* from the coal mining site at Ostrava, Czech Republic, belongs to the cosmopolitan species *G. sulphuraria*, for now the only eukaryotic organism forming visible biomass on a burning coal-waste heap [10]. This was the first evidence of this species growing in central Europe, and isolates were closely related to the Italian strains, together forming the continental European lineage of *G. sulphuraria* [21]*.* Another, nonthermophilic strain of *Galdieria*, also found in the Czech Republic [22], referred to as CCALA 965 (Culture Collection of Autotrophic Organisms, Institute of Botany, CAS, Třeboň, Czech Republic) was found to belong to the species *G. phlegrea*, so far known only from Italy [13, 14].

### **3. Morphology and extremophile properties**

Morphology of the unicellular taxa Cyanidiales is relatively simple. Thick-walled cells are of a spherical shape and usually contain one chloroplast, 1–3 mitochondria, a nucleus, a vacuole, and energy reserve products [8, 12, 15, 23, 24].

Representatives of the order Cyanidiales are unparalleled among phototrophic microorganisms (eukaryotes) in their ability to thrive in acidic (pH 0.5–3.5) and high temperature (38–56°C) geothermal environments. Soils, sediments, and endolithic habitats around hot springs, boiling mud pools, and steaming fumaroles are typical for these extremophiles, which are dominant in local microbial communities [5, 9, 10, 14, 17–20, 25–30]. They are the principal photosynthetic organisms found in hot acidic waters [31], where even photosynthetic prokaryotes, such as the cyanobacteria, are completely absent [32–34].

*G. sulphuraria* is a unicellular, spherical, spore-forming red alga. In addition to acidophilic and thermophilic properties, it has the ability to grow phototrophically, mixotrophically and moreover heterotrophically while utilizing sugars, alcohols or amino acids [7, 35, 36]. *G. sulphuraria* cells are morphologically indiscernible from cells of *Cyanidium caldarium*, however, are well recognizable thanks to their ready ability to grow heterotrophically in the dark [16, 18]. In its natural environment, *G. sulphuraria* has a yellow to green color; however, when heterotrophically grown in liquid medium, it looks like yellow-green to dark blue-green. The cell size of *G. sulphuraria* is larger than that of *C. caldarium*. It reproduces by endospore formation ranging from four to thirty-two. As originally described, *G. sulphuraria* has a single, cup-shaped, parietally localized chloroplast [8] and includes a vacuole and mitochondria [11].

Morphological similarities between *G. sulphuraria* and *G. phlegrea* are so high that methods for recognition of these species, their habitats, and growth requirements, together with molecular analyses, are used.

*G. phlegrea* (**Figure 1**) prefers a relatively low temperature (25–38°C) for growth and inhabits rather dry endolithic sites with high acidity (pH 0.5–1.5) [9, 14, 22]. The Latin word *phlegreus* means volcanic, what is consistent with the specific locality, (Campi Flegrei, Naples, Italy), where the alga was found [14]. The locality provides diverse environmental conditions in the form of hot springs, streams, mud, rock walls, with different pHs and temperature ranges, producing the different microhabitats occurring in that site. [14].

Strain DB01 of *G. phlegrea* from the Tinto River (Spain) has typical coccoid cells with thick smooth cell walls. Mature cells reach the average size of 6.4 μm. The cell possesses a blue-green chloroplast without pyrenoids. Typical for the alga is asexual

*Microalgae - From Physiology to Application*

concentration of lipids.

**2. Taxonomy and biodiversity**

thermo-acidotolerancy of this lineage [9].

*Cyanidioschyzon merolae* [5, 9, 10, 12, 16].

application in biotechnology [3]. The autotrophic cultivation of *Galdieria* follows predominant research trends in microalgal pigments, ß carotene, astaxanthin, and phycocyanin used in feed, in foods, in health applications or biofuels production. The composition of storage glycogen and lipids for biofuels can be largely changed depending on the growth conditions. *Galdieria* biomass has potential for use as food ingredients, both for protein-rich or insoluble dietary fiber-rich diets and for its low

The order Cyanidiales from the class Cyanidiophyceae are a group of asexual, unicellular organisms that diverged from ancestral red algae around 1.3 billion years ago [4, 5]. These unicellular red algae were classified into three genera, *Cyanidium*, *Cyanidioschyzon*, and *Galdieria*. *Cyanidioschyzon merolae* is clearly recognizable thanks to its characteristic size and shape, but the other two algae are morphologically very similar. Until 1981 *Cyanidium caldarium* was used as a synonym for *Galdieria sulphuraria*, however, this species can grow only autotrophically while *G. sulphuraria* is also able to grow heterotrophically [6, 7]. Based on morphological, ultrastructural and ecophysiological studies the class Cyanidiophyceae was therefore instituted, containing the family Cyanidiaceae, including *Cyanidium caldarium*

Different Cyanidiophycean species, including *Galdieria*, are found all over the world; however, their distribution is discontinuous as they are restricted to hot springs and geothermal habitats. This is related to the discontinuity of geothermal environments. Two decades ago, little was known about their biodiversity, their population structures, and the phylogenetic relationships of Cyanidiales.

Research based on environmental PCR studies revealed an unexpected level of genetic diversity among Cyanidiales. It was demonstrated that the Cyanidiales comprise a species-rich branch of red algae [9]. The high divergence rates in the Cyanidiales could be possibly explained by an elevated mutation rate in these taxa, resulting potentially from DNA damage in their extreme environments. The analyses also reject the putative mesophilic origin of Cyanidiales and suggest ancestral

Sequencing of the *rbcL* gene with high sequence divergence within the genus has contributed to the taxonomy of *Galdieria* [10–12]. A cladogram defining molecular relationships among these algae shows that *Cyanidium caldarium* and *Cyanidioschyzon merolae* form a sister group relationship with *Galdieria* [11]. The genus *Galdieria* is divided into two clades, one of which includes *G. sulphuraria* accessions from Naples (Italy), California, and Yellowstone and the other one includes *G. sulphuraria* accessions from Java (Indonesia) and Russian species [11]. Based on molecular phylogenetics, three well supported *Galdieria* species exist: *G. maxima* Sentsova, *G. sulphuraria* (Galdieri) Merola (including two Russian species *G. partita* Sentsova and *G. daedala* Sentsova, described based on morphological

Consequently, the main lineages were identified: *G. phlegrea* [14] comprising strains thriving in acidic non-thermophilic Italian sites; *G. sulphuraria*, a group that is geographically dispersed worldwide, including *G. sulphuraria* strains as well as *G. partita* and *G. daedala*, isolated from acid-thermal springs in Russia [15]. A final lineage contains the cosmopolitan species *G. maxima* [15], which is clustered independently from the *Galdieria* clade (*G. sulphuraria* + *G. phlegrea*) and shows an unexplained sister group relationship with the morphologically distinct

differences) and *G. phlegrea* Pinto, Ciniglia, Cascone et Pollio [9, 13, 14].

and the family Galdieriaceae, including *Galdieria sulphuraria* [8].

**106**

#### **Figure 1.**

*Microphotographs of* Galdieria phlegrea *in bright field (A) and fluorescence (B). Nuclei in the panel B are in blue (stained by DAPI) and chloroplasts are in red (autofluorescence). The chloroplasts of large mother dividing cells are not visible. The bar is 10 μm.*

reproduction by autospores originating in the parental cell and resulting in autosporangia with 2–8 daughter cells. Testing the culture conditions of the isolate DB01 showed that the algae were not strictly thermophilic [13].

*G. maxima* is characterized by facultative heterotrophy; however, *G. maxima* strains grow very poorly when cultivated under dark conditions. Spherical cells are significantly larger (10–16 μm diameter) compared with other thermoacidophilic algal species [18, 23]. Cell size is thus used as the main character to distinguish one from the other. Inside the cell are at least two parietal plastids, lobe or oval shaped [37].

### **4. Genomes**

Genetic information for the red algae *Galdieria* (*G. maxima, G. partita*, and *G. sulphuraria*) is located in the nucleus, in two small chromosomes, which differ in length. The smaller chromosome ranges from 0.8 to 1.8 μm and the larger one from 1.2 to 2.3 μm. The genome is characterized by an unusually high gene density, small or absent introns, and very few repetitive sequences. A genome size of 10.8 Mbp was estimated for *G. sulphuraria* [38]. In other strains of *G. sulphuraria*, genome sizes were found between 9.8 and 14.2 Mbp [39]. These genome characteristics refer to the smallest known genomes of all free living eukaryotes [38, 40].

The mitochondrial genome is extremely small in size with a very low genetic content. It is characterized by the highest guanine-cytosine content among all red algae.

The plastid genome contains a large number of intergenic stem-loop structures but is otherwise rather typical in size, structure, and content in comparison with other red algae. It is assumed that the unique genomic characteristic resulted from both the harsh conditions in which *Galdieria* lives and its unusual ability to grow mixotrophically, heterotrophically, and endolithically. The authors [41] suggested that "these conditions place additional mutational pressures on the mitogenome due to the increased reliance on the mitochondrion for energy production, whereas the decreased reliance on photosynthesis and the presence of numerous stem-loop structures may shield the plastome from similar genomic stress."

**109**

*The Red Microalga* Galdieria *as a Promising Organism for Applications in Biotechnology*

*Galdieria* with its extensive extremophilic properties, which are unique not only among all eukaryotic organisms but even in extremophilic prokaryotes, has a broad utilization in biotechnology. It is the only algae that can grow photo-, mixo-, and heterotrophically to biomass concentrations above 100 g/L dry weight [42]. For heterotrophic growth, it can use over 27 different kinds of sugars and polyols to produce a huge biomass and beneficial compounds [7, 43, 44]. It tolerates concentrations of glucose and fructose up to 166 g/L, salt concentrations up to 1–2 M, and

flow cultures [43]. The ability of *Galdieria* to grow under conditions intolerable for other organisms, even prokaryotic ones, predetermines its biotechnical applications in such surroundings as different, often toxic, wastewaters, treatment of acid mine drainage, selective metal precipitation, bioremediation of acidic metal-contaminated areas, or recovery of critical and scarce metals from secondary sources.

Recycling of valuable components and nutrients from wastewaters using algae has recently been studied extensively. But only limited types of wastewaters can be treated because wastewaters are generally acidic and most algal species grow with difficulty at low pH, and absorption rates that can be achieved by bioaccumulation decrease substantially [46–49]. The acidophilic alga *G. sulphuraria* is the only alga that has commercial potential for remediation of these wastewaters [50, 51]. Nutrient removal from municipal wastewater by the alga *G. sulphurea* was found to be very efficient for ammoniacal-nitrogen (88.3%) and phosphate (95.5%) in large scale outdoor bioreactors [51]. Additionally, many crucial elements, including phosphate and rare earth elements from wastewater were successfully bio-sorbed [51, 52]. It can be concluded that *G. sulphuraria* can be applied as a preferred strain for energy-efficient nutrient removal from urban wastewaters [51], achieving higher nutrient removal efficiencies and removal rates than other strains. This alga can also be used for bio-sorption of precious metals from metal-containing wastewaters. The great advantage is that precious metals can be efficiently bio-sorbed by *Galdieria* cells even if they are present in very low concentrations. Over 90% of gold and palladium were recovered from aqua-regia-based metallic wastewater where metal concentrations were so low that they could not be recycled chemically or pyro-metallurgically. Because the entire process could be completed within 1 h, the use of *G. sulphuraria* has promising applications in metal recovery [53], particularly where Pt and Au could be selectively re-eluted from cells into a solution containing

biomass in continuous

*DOI: http://dx.doi.org/10.5772/intechopen.89810*

pH values below pH 1 [42, 45] and can attain 80–110 g L<sup>−</sup><sup>1</sup>

0.2 M ammonium salts without other contaminating metals [54].

Lanthanides [Rare Earth Elements (REEs)] have unique magnetic and catalytic properties and are, up to now, irreplaceable materials in numerous technologies, for example, wind turbines, solar panels, batteries, fluorescent lamps, computer and mobile monitors, TV screens etc. They are also used as fertilizers in agriculture, in

Methods for extraction of lanthanides from ores, including pyro-metallurgy and hydro-metallurgy, have severe negative environmental impacts, as well as being expensive. Currently, industrial extraction of lanthanides from monazite involves either a basic process that uses concentrated sodium hydroxide or an acidic process that uses concentrated sulfuric acid. These processes generate large amounts of

**5. Biotechnological applications**

**5.1 Wastewaters**

**5.2 Rare earth elements**

aquaculture, or as animal growth enhancers.

*The Red Microalga* Galdieria *as a Promising Organism for Applications in Biotechnology DOI: http://dx.doi.org/10.5772/intechopen.89810*
