**5. Biotechnological applications**

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

#### **5.1 Wastewaters**

*Microalgae - From Physiology to Application*

*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

*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* 

*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

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

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

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

to the smallest known genomes of all free living eukaryotes [38, 40].

structures may shield the plastome from similar genomic stress."

showed that the algae were not strictly thermophilic [13].

**108**

algae.

oval shaped [37].

**4. Genomes**

**Figure 1.**

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

#### **5.2 Rare earth elements**

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 aquaculture, or as animal growth enhancers.

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

hazardous waste containing thorium and uranium [55]. Moreover, requirements for REEs are continually increasing, becoming critical due to risks of reduced availability of resources and their possible exhaustion.

One way to solve the problem would be efficient, sustainable, and cheap recycling of REE-containing wastewaters and others industrial wastes.

Considerable research efforts have been directed toward the development of efficient biological methods for recovering small amounts of these materials from wastewater systems [48, 49]. Research has recently focused on environmentally friendly technologies of metal recovery, including REEs, from secondary resources [56, 57] including bio-sorption by algae or cyanobacteria [52, 58]; for review, see [59, 60].

However, if REEs were present in an aquatic environment, together with other metals, most algae could not accumulate high concentrations of REEs [61, 62] due to metal-inhibited growth. The extraction of REEs or other metals have now been simplified by the use of *Galdieria* cells, which were effective in the recovery of many crucial elements, including phosphates and REEs [51, 52].

Similar to its relative *Cyanidium caldarium, G. sulphuraria* is resistant to high concentrations of metals in solution, including Al3+. Moreover, it could be used to selectively recover lanthanides and Cu2+ ions from water containing various kinds of metals at a pH of 2.5. The concentration of soluble metals in solution remained unchanged at pH values within the range 0.5–5.0 [52]. In contrast, this process is usually difficult to achieve by bacterial bio-sorption. Lowering of pH to 1.0–1.5 enabled the recovery of lanthanides from cells whereas Cu2+ ions remained dissolved in aqueous acid. The use of *G. sulphuraria* also allowed recovery of over 90% of low levels of metals (0.5 ppm) from solution by cell fractionation at pH values in the range of 1.5–2.5. This system did not require any genetic manipulation or treatment of the cells for the efficient recovery of lanthanides [52].

Recycling from different mineral ores and electronic wastes (luminophores) often meets difficulties in that REEs are not suitable for bio-sorption because they are present in solid forms and are almost insoluble in nutrient solutions for algal cultivation. The material can be readily dissolved in aqueous acid, but the efficiency of metal bio-sorption for most algae is usually decreased under acidic conditions or the algae cannot grow at a low pH. Application of extremophilic red alga *Galdieria* would therefore be an advantageous solution to this problem and seems to be the aim of future research. The species *Galdieria phlegrea* has already been used to test the bio-accumulation of REEs from luminophores added into the medium in the form of a powder. Algal cells were cultured mixotrophically in a liquid medium with the addition of glycerol as a source of carbon. Luminophores from two different sources (fluorescence lamps and energy saving light bulbs) were tested. In spite of the low solubility of luminophores, *G. phlegrea* could grow in the presence of luminophores and accumulate REEs [63, 64].

Another rich source of lanthanides is bauxite residue, called red mud, which is a by-product of the production of alumina (aluminum oxide) from bauxite. However, less than 2% of the residue produced annually is currently being reused [65], due to difficulties related to high pH, salinity, low solid content, size of fine particles, and the leaching of metals [66]. The ability to grow in the presence of red mud and accumulate REEs was successfully tested with *G. phlegrea* (**Figure 2**) [67].

To conclude, the alga *G. sulphuraria* offers great potential for the direct recovery of REEs from metal-containing wastewaters (even if present at very low concentrations) or from solid waste material (luminophores) as well as for bio-mining from bauxite ore residue (red mud).

**111**

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

Phycocyanin is an important compound that can be obtained from microalgal and cyanobacterial cultures. It is used as a fluorescent marker in diagnostic histochemistry [68, 69] and as a dye in foods and cosmetics [70] or as a therapeutic agent [71, 72]. Production of phycocyanin as a photosynthetic pigment in most microalgae grown heterotrophically is low and not suitable for biotechnological applications [73]. In contrast, phycocyanin as a major pigment of *G. sulphuraria* can be produced even under heterotrophic conditions in darkness [7, 74]. Due to superior biomass productivity, the productivity of phycocyanin in cultures of *G. sulphuraria* was 1–2 orders of magnitude greater than in *Arthrospira* (*Spirulina*) *platensis*, which was used recently for commercial production of phycocyanin [43] and was dependent on sunlight and climatic conditions. Besides light independence, *G. sulphuraria* tolerates a concentrated medium and can metabolize many different compounds as

*Electron microphotograph of the dense freeze-dried culture of* Galdieria phlegrea *used for REEs recovery. The* 

For example, *G. sulphuraria* could grow in restaurant and bakery waste hydrolysates in which sugars and free amino acids were utilized as substrates. Ammonium and inorganic nutrients were, however, needed in order to maximize phycocyanin

The feasibility of utilizing crude glycerol (a major waste by-product of biofuel production from oilseed rape) as a carbon source for heterotrophic growth of green

*G. sulphuraria* has also been grown on sugar beet molasses [42]. Under heterotrophic conditions, phycocyanin synthesis depends mostly on available ammonium ions [77]. Ammonium sulphate was tolerated in higher molar concentrations than

*G. sulphuraria* is well suited for heterotrophic growth to an extremely high cell density, which is among the highest biomass concentrations ever reported for microalgal cultures. Nearly 5% of sugar is employed for biomass yield, which is comparable to the biomass yields in industrially important heterotrophic microor-

The high tolerance of *Galdieria* species to concentrated substances is probably an adaptation to the high concentrations of sulfuric acid and other salts present

microalgae [54] was confirmed for *Chlorella* and also for *G. sulphuraria*.

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

**5.3 Phycocyanin**

**Figure 2.**

synthesis [76].

ganisms [78].

a source of carbon and energy [7, 75].

*bar is 10 μm. (provided by Dr. Jens Hartmann).*

glucose, fructose, or sodium chloride [22, 23].

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

#### **Figure 2.**

*Microalgae - From Physiology to Application*

review, see [59, 60].

lanthanides [52].

luminophores and accumulate REEs [63, 64].

bauxite ore residue (red mud).

ity of resources and their possible exhaustion.

hazardous waste containing thorium and uranium [55]. Moreover, requirements for REEs are continually increasing, becoming critical due to risks of reduced availabil-

One way to solve the problem would be efficient, sustainable, and cheap recy-

Considerable research efforts have been directed toward the development of efficient biological methods for recovering small amounts of these materials from wastewater systems [48, 49]. Research has recently focused on environmentally friendly technologies of metal recovery, including REEs, from secondary resources [56, 57] including bio-sorption by algae or cyanobacteria [52, 58]; for

However, if REEs were present in an aquatic environment, together with other metals, most algae could not accumulate high concentrations of REEs [61, 62] due to metal-inhibited growth. The extraction of REEs or other metals have now been simplified by the use of *Galdieria* cells, which were effective in the recovery of

Similar to its relative *Cyanidium caldarium, G. sulphuraria* is resistant to high concentrations of metals in solution, including Al3+. Moreover, it could be used to selectively recover lanthanides and Cu2+ ions from water containing various kinds of metals at a pH of 2.5. The concentration of soluble metals in solution remained unchanged at pH values within the range 0.5–5.0 [52]. In contrast, this process is usually difficult to achieve by bacterial bio-sorption. Lowering of pH to 1.0–1.5 enabled the recovery of lanthanides from cells whereas Cu2+ ions remained dissolved in aqueous acid. The use of *G. sulphuraria* also allowed recovery of over 90% of low levels of metals (0.5 ppm) from solution by cell fractionation at pH values in the range of 1.5–2.5. This system did not require any genetic manipulation or treatment of the cells for the efficient recovery of

Recycling from different mineral ores and electronic wastes (luminophores) often meets difficulties in that REEs are not suitable for bio-sorption because they are present in solid forms and are almost insoluble in nutrient solutions for algal cultivation. The material can be readily dissolved in aqueous acid, but the efficiency of metal bio-sorption for most algae is usually decreased under acidic conditions or the algae cannot grow at a low pH. Application of extremophilic red alga *Galdieria* would therefore be an advantageous solution to this problem and seems to be the aim of future research. The species *Galdieria phlegrea* has already been used to test the bio-accumulation of REEs from luminophores added into the medium in the form of a powder. Algal cells were cultured mixotrophically in a liquid medium with the addition of glycerol as a source of carbon. Luminophores from two different sources (fluorescence lamps and energy saving light bulbs) were tested. In spite of the low solubility of luminophores, *G. phlegrea* could grow in the presence of

Another rich source of lanthanides is bauxite residue, called red mud, which is a by-product of the production of alumina (aluminum oxide) from bauxite. However, less than 2% of the residue produced annually is currently being reused [65], due to difficulties related to high pH, salinity, low solid content, size of fine particles, and the leaching of metals [66]. The ability to grow in the presence of red mud and

To conclude, the alga *G. sulphuraria* offers great potential for the direct recovery of REEs from metal-containing wastewaters (even if present at very low concentrations) or from solid waste material (luminophores) as well as for bio-mining from

accumulate REEs was successfully tested with *G. phlegrea* (**Figure 2**) [67].

cling of REE-containing wastewaters and others industrial wastes.

many crucial elements, including phosphates and REEs [51, 52].

**110**

*Electron microphotograph of the dense freeze-dried culture of* Galdieria phlegrea *used for REEs recovery. The bar is 10 μm. (provided by Dr. Jens Hartmann).*

#### **5.3 Phycocyanin**

Phycocyanin is an important compound that can be obtained from microalgal and cyanobacterial cultures. It is used as a fluorescent marker in diagnostic histochemistry [68, 69] and as a dye in foods and cosmetics [70] or as a therapeutic agent [71, 72]. Production of phycocyanin as a photosynthetic pigment in most microalgae grown heterotrophically is low and not suitable for biotechnological applications [73]. In contrast, phycocyanin as a major pigment of *G. sulphuraria* can be produced even under heterotrophic conditions in darkness [7, 74]. Due to superior biomass productivity, the productivity of phycocyanin in cultures of *G. sulphuraria* was 1–2 orders of magnitude greater than in *Arthrospira* (*Spirulina*) *platensis*, which was used recently for commercial production of phycocyanin [43] and was dependent on sunlight and climatic conditions. Besides light independence, *G. sulphuraria* tolerates a concentrated medium and can metabolize many different compounds as a source of carbon and energy [7, 75].

For example, *G. sulphuraria* could grow in restaurant and bakery waste hydrolysates in which sugars and free amino acids were utilized as substrates. Ammonium and inorganic nutrients were, however, needed in order to maximize phycocyanin synthesis [76].

The feasibility of utilizing crude glycerol (a major waste by-product of biofuel production from oilseed rape) as a carbon source for heterotrophic growth of green microalgae [54] was confirmed for *Chlorella* and also for *G. sulphuraria*.

*G. sulphuraria* has also been grown on sugar beet molasses [42]. Under heterotrophic conditions, phycocyanin synthesis depends mostly on available ammonium ions [77]. Ammonium sulphate was tolerated in higher molar concentrations than glucose, fructose, or sodium chloride [22, 23].

*G. sulphuraria* is well suited for heterotrophic growth to an extremely high cell density, which is among the highest biomass concentrations ever reported for microalgal cultures. Nearly 5% of sugar is employed for biomass yield, which is comparable to the biomass yields in industrially important heterotrophic microorganisms [78].

The high tolerance of *Galdieria* species to concentrated substances is probably an adaptation to the high concentrations of sulfuric acid and other salts present

in acidic springs. *G. sulphuraria* tolerated and grew well concentrations of glucose and fructose of up to 166 g/L (0.9 M) and an ammonium sulphate concentration of 22 g/L (0.17 M) without negative effects on specific growth rate. In carbon-limited fed-batch cultures, biomass dry weight concentrations of 80–120 g/L were obtained while phycocyanin accumulated to concentrations between 250 and 400 mg/L [42].

The ability of *G. sulphuraria* to accumulate high levels of phycocyanin in heterotrophic or mixotrophic cultures compete with or at least represents an alternative to the cyanobacterium, *Arthrospira (Spirulina) platensis* that is currently used for synthesis of phycocyanin [77].

Since a number of positive health effects have been associated with phycocyanin [79], and phycocyanin from *A. platensis* has been approved for food use in the USA and EU in 2013 and 2014, respectively, interests in applications of phycocyanin have increased substantially over recent years [80].

In addition to phycocyanin, *G. sulphuraria* could also provide floridosides suggested as a commercial products [81–83]. Its biomass was also tested and found to be a suitable and safe component in foods, as well as a dietary supplement [84].

#### **5.4 Biofuels**

The world-wide and continuous increase in fossil fuel consumption, leading probably in the relatively near future to an exhaustion of resources, has led to increased research for alternative energy sources. Production of biofuels by algae might provide a viable alternative to fossil fuels; however, this technology must overcome a number of serious obstacles before it could compete in the fuel market and be broadly deployed. Application of remarkably extremophilic *G. sulphuraria* could overcome at least some of these.

Microalgae often become contaminated with other microorganisms in largescale outdoor cultivations, which is a major problem that inhibits algal growth and decreases the quality of biofuel and high-value products. A lack of resistance to these factors could be catastrophic for future algae farmers. The red alga *G. sulphuraria* and other species of the same genus have great potential to produce large quantities of biofuel [53] and other beneficial compounds without becoming contaminated with other microorganisms, under both mixotrophic and heterotrophic conditions. Furthermore, the algae are tolerant of pH and temperature extremes that offer a reliable means of controlling the composition of large-scale cultures.

#### **5.5 Glycogen**

The extremophilic red algae, similarly to other Rhodophyta, produce glycogen as energy and carbon reserves, instead of starch, which is characteristic of other microalgae and higher plants [81]. Glycogen, in contrast to starch, is readily soluble in cold water and more accessible by enzymes. In red algae, glycogen accumulates in a lower molecular weight form than glycogen from other microalgae and is a highly branched (higher than any other glycogen) glucose polymer [81]. Amylopectin, as a highly branched glucose polymer in starch, is used in various products such as peritoneal dialysis solutions and sports drinks. However, it is costly to prepare because of its insoluble, granular nature. The application of glycogen offers a cheaper alternative.

The alga *G. sulphuraria* can grow to a very high biomass concentration [42], accumulating glycogen up to 50% of the dry cell weight. Another advantage of this alga is that it can grow heterotrophically using many organic sources and also very cheap waste glycerol [84].

**113**

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

Apart from *G. sulphuraria*, the production of glycogen by most other microorganisms is too low for biotechnological applications because it is produced only under growth limiting conditions [85]. Thus, *G. sulphuraria* can be used as a cheap and efficient producer of glycogen, which could be applied as an alternative to

Large amounts of glycogen were obtained in mixotrophic cultures, [53] where the maximum glycogen content per mL of culture was almost 10- and 2-fold greater than those of autotrophic and heterotrophic cultures, respectively. The accumulation of glycogen was enhanced by the addition of glucose, and the amount and composition of glycogen were determined by growth conditions. It is assumed that in addition to glycogen, other forms of carbon may be stored, although pathways

Because of their high content of protein, algal biomass, in general, and green algae particularly, have been used in many foods, mostly in the form of dried

Difficulties in introducing microalgal-based ingredients into foods are technological and include sensorial obstacles such as its unattractive green-brownish color and unpleasant fishy smell increasing after longer storing [87]. Another problem is bacterial contamination, which decreases the commercial quality of algal biomass. Such disadvantages of green algae are not encountered using *Galdieria* species. *G. sulphuraria* can grow heterotrophically even in large-scale bioreactors under so extreme conditions that contamination by other organisms is not likely. Biomass is colorless, has a low lipid content, mainly of monounsaturated fatty acids, and oxidation during shelf life is negligible. Heterotrophic growth enables high cell densities to be achieved using cheap glycerol as a source of carbon. Consequently, in addition to other specific applications, red algal biomass can be used as a source of protein and other macronutrients. *G. sulphuraria* is rich in proteins (26–32%) and polysaccharides (63–69%), and poor in lipids. Under heterotrophic cultivation conditions, the lipid moiety mainly contained monounsaturated fatty acids. Nutritional applications of red algae were firstly suggested by Bailey and Staehelin [87], who found very high levels of protein in

*G. sulphuraria* proteins are strictly associated with polysaccharide components and therefore not digestible. However, a commercial enzyme preparation containing a mixture of polysaccharidases was developed, and *G. sulphuraria* proteins were good substrates for human gastrointestinal enzymes. *G. sulphuraria* biomass therefore has the potential to be used either for protein-rich or for insoluble dietary fiber-rich applications. Among micronutrients, some B group vitamins and pigments are present. Carotenoids are minor pigments in *G. sulphuraria*, detected only in the autotrophic algae, the main ones being astaxanthin and lutein. The absence of carotenoids under heterotrophic growth conditions is due to the lack of photosynthesis. Phycobiliproteins are present under heterotrophic and mixotrophic cultivation conditions. The cells grown on organic source of carbon frequently lose their photosynthetic antenna undermining the accumulation of the phycobilins. In *G. sulphuraria*, allophycocyanin is the dominant form in the autotrophic algae, while phycoerithrin was the main phycobiliprotein in the

*G. sulphuraria* can therefore be used to develop new food ingredients, including

preparations that are rich in bioavailable proteins and dietary fiber [84].

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

starch in several fields [83].

are, as yet, unknown [75, 86].

**5.6 Nutritional applications**

biomass.

their cell walls.

heterotrophic algae.

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

Apart from *G. sulphuraria*, the production of glycogen by most other microorganisms is too low for biotechnological applications because it is produced only under growth limiting conditions [85]. Thus, *G. sulphuraria* can be used as a cheap and efficient producer of glycogen, which could be applied as an alternative to starch in several fields [83].

Large amounts of glycogen were obtained in mixotrophic cultures, [53] where the maximum glycogen content per mL of culture was almost 10- and 2-fold greater than those of autotrophic and heterotrophic cultures, respectively. The accumulation of glycogen was enhanced by the addition of glucose, and the amount and composition of glycogen were determined by growth conditions. It is assumed that in addition to glycogen, other forms of carbon may be stored, although pathways are, as yet, unknown [75, 86].

#### **5.6 Nutritional applications**

*Microalgae - From Physiology to Application*

synthesis of phycocyanin [77].

**5.4 Biofuels**

**5.5 Glycogen**

cheaper alternative.

cheap waste glycerol [84].

increased substantially over recent years [80].

could overcome at least some of these.

in acidic springs. *G. sulphuraria* tolerated and grew well concentrations of glucose and fructose of up to 166 g/L (0.9 M) and an ammonium sulphate concentration of 22 g/L (0.17 M) without negative effects on specific growth rate. In carbon-limited fed-batch cultures, biomass dry weight concentrations of 80–120 g/L were obtained while phycocyanin accumulated to concentrations between 250 and 400 mg/L [42]. The ability of *G. sulphuraria* to accumulate high levels of phycocyanin in heterotrophic or mixotrophic cultures compete with or at least represents an alternative to the cyanobacterium, *Arthrospira (Spirulina) platensis* that is currently used for

Since a number of positive health effects have been associated with phycocyanin [79], and phycocyanin from *A. platensis* has been approved for food use in the USA and EU in 2013 and 2014, respectively, interests in applications of phycocyanin have

In addition to phycocyanin, *G. sulphuraria* could also provide floridosides suggested as a commercial products [81–83]. Its biomass was also tested and found to be a suitable and safe component in foods, as well as a dietary supplement [84].

The world-wide and continuous increase in fossil fuel consumption, leading probably in the relatively near future to an exhaustion of resources, has led to increased research for alternative energy sources. Production of biofuels by algae might provide a viable alternative to fossil fuels; however, this technology must overcome a number of serious obstacles before it could compete in the fuel market and be broadly deployed. Application of remarkably extremophilic *G. sulphuraria*

Microalgae often become contaminated with other microorganisms in largescale outdoor cultivations, which is a major problem that inhibits algal growth and decreases the quality of biofuel and high-value products. A lack of resistance to these factors could be catastrophic for future algae farmers. The red alga *G. sulphuraria* and other species of the same genus have great potential to produce large quantities of biofuel [53] and other beneficial compounds without becoming contaminated with other microorganisms, under both mixotrophic and heterotrophic conditions. Furthermore, the algae are tolerant of pH and temperature extremes that offer a reliable means of controlling the composition of large-scale cultures.

The extremophilic red algae, similarly to other Rhodophyta, produce glycogen as energy and carbon reserves, instead of starch, which is characteristic of other microalgae and higher plants [81]. Glycogen, in contrast to starch, is readily soluble in cold water and more accessible by enzymes. In red algae, glycogen accumulates in a lower molecular weight form than glycogen from other microalgae and is a highly branched (higher than any other glycogen) glucose polymer [81]. Amylopectin, as a highly branched glucose polymer in starch, is used in various products such as peritoneal dialysis solutions and sports drinks. However, it is costly to prepare because of its insoluble, granular nature. The application of glycogen offers a

The alga *G. sulphuraria* can grow to a very high biomass concentration [42], accumulating glycogen up to 50% of the dry cell weight. Another advantage of this alga is that it can grow heterotrophically using many organic sources and also very

**112**

Because of their high content of protein, algal biomass, in general, and green algae particularly, have been used in many foods, mostly in the form of dried biomass.

Difficulties in introducing microalgal-based ingredients into foods are technological and include sensorial obstacles such as its unattractive green-brownish color and unpleasant fishy smell increasing after longer storing [87]. Another problem is bacterial contamination, which decreases the commercial quality of algal biomass. Such disadvantages of green algae are not encountered using *Galdieria* species. *G. sulphuraria* can grow heterotrophically even in large-scale bioreactors under so extreme conditions that contamination by other organisms is not likely. Biomass is colorless, has a low lipid content, mainly of monounsaturated fatty acids, and oxidation during shelf life is negligible. Heterotrophic growth enables high cell densities to be achieved using cheap glycerol as a source of carbon. Consequently, in addition to other specific applications, red algal biomass can be used as a source of protein and other macronutrients. *G. sulphuraria* is rich in proteins (26–32%) and polysaccharides (63–69%), and poor in lipids. Under heterotrophic cultivation conditions, the lipid moiety mainly contained monounsaturated fatty acids. Nutritional applications of red algae were firstly suggested by Bailey and Staehelin [87], who found very high levels of protein in their cell walls.

*G. sulphuraria* proteins are strictly associated with polysaccharide components and therefore not digestible. However, a commercial enzyme preparation containing a mixture of polysaccharidases was developed, and *G. sulphuraria* proteins were good substrates for human gastrointestinal enzymes. *G. sulphuraria* biomass therefore has the potential to be used either for protein-rich or for insoluble dietary fiber-rich applications. Among micronutrients, some B group vitamins and pigments are present. Carotenoids are minor pigments in *G. sulphuraria*, detected only in the autotrophic algae, the main ones being astaxanthin and lutein. The absence of carotenoids under heterotrophic growth conditions is due to the lack of photosynthesis. Phycobiliproteins are present under heterotrophic and mixotrophic cultivation conditions. The cells grown on organic source of carbon frequently lose their photosynthetic antenna undermining the accumulation of the phycobilins. In *G. sulphuraria*, allophycocyanin is the dominant form in the autotrophic algae, while phycoerithrin was the main phycobiliprotein in the heterotrophic algae.

*G. sulphuraria* can therefore be used to develop new food ingredients, including preparations that are rich in bioavailable proteins and dietary fiber [84].
