1. Types and Chemistry of Carotenoids
Carotenoids are chemical compounds belonging to the group of terpenoids. Terpenoids are also called isoprenoids because their structure consists of eight units of isoprene (C
5H
8). There are two categories of carotenoids, both with biotechnological interest; the carotenes consist of hydrocarbons, and the xanthophylls with their oxygenated derivatives
[1].
Figure 1 presents the chemical structures of the most common carotenes and xanthophylls produced by microorganisms. Carotenes include carotenoids such as
α-,
β- and
γ-carotene, lycopene, torulene, neurosporene and others. The group of xanthophylls includes astaxanthin, zeaxanthin, lutein, torularhodin, canthaxanthin, violaxanthin and others.
Figure 1. Chemical structures of the most important carotenoids produced biotechnologically.
Natural carotenoids are biosynthesized via two major pathways, namely the 2-C-methyl-D-erythritol 4-phosphate (MEP) and the mevalonate (MVA) pathways. The two main terpenoid precursors generated from these pathways are dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), respectively. Geranylgeranyl diphosphate (GGPP) is then generated from IPP isomerization, carried out by the addition of three IPP molecules to DMAPP, which is subsequently condensed to phytoene
[2][3]. Finally, phytoene, which is the first carotenoid synthesized from the pathways, is desaturated and isomerized to lycopene
[3][4]. The structure of all carotenoids can be derived from the structure of acyclic C
40H
56 (corresponds to the structure of lycopene) via hydrogenation, dehydrogenation, cyclization or oxidation
[1]. The main characteristic of the carotenoid structure is the long hydrocarbon chain, consisting of conjugated double bonds. The delocalized π electrons from the conjugated double bonds system are the reason that carotenoids exhibit high antioxidant properties. In β-carotene, both ends are cyclized, while lycopene is characterized by two acyclic parts in its structure. The number of conjugated double bonds is also linked to the color of the carotenoid
[5]. Molecules with a high number of conjugated double bonds absorb at higher wavelengths, resulting in a yellow-red color. A characteristic example is the formation of lycopene during the maturation of tomatoes from phytoene by phytoene desaturase. The conjugated bonds in the carotenoids’ structure have the ability to absorb visible light and are thus responsible for the molecule’s color. Phytoene is a colorless compound with three conjugated double bonds that is converted to phytofuene (pale yellow) with five conjugated double bonds,
ζ-carotene (yellow) with seven conjugated double bonds, neurosporene (orange) with nine conjugated double bonds and finally to lycopene (red) with eleven conjugated double bonds
[6].
The number of conjugated bonds in the molecule determines the color and the absorbance maxima. For instance, lycopene is an acyclic molecule of red color and because of the 11 conjugated double bonds, its maximum absorbance is located at 444, 470 and 502 nm. On the other hand, α-carotene, with 10 conjugated double bonds (one of them in the cyclic part), absorbs at 422, 445 and 473 nm, demonstrating an orange color
[5].
The position of the side groups linked to the atoms where the double bonds are located divides carotenoids into
trans or
cis isomers.
Trans carotenoids are also characterized as
all-E and
cis carotenoids as
Z [7]. The
all-E characterization refers to a structure where all double bonds are in
trans formation, while the
Z one differs depending on the double bond that is found in
cis formation.
Figure 2 illustrates the
all-E isomer (
trans) of lycopene and one of its
cis isomers. Carotenoids occur predominantly in their
all-E form; however the extent of antioxidant activities and bioavailability in the human body is highly dependent on the type of carotenoid under investigation
[8]. Many studies evidence that
Z-isomers (especially in the case of lycopene and astaxanthin) exhibit higher scavenging activities than their
trans counterparts, while also presenting greater bioavailability
[8][9][10]. Likewise, regarding their abundance, there is a wide distribution of carotenoid isomers in nature. For example, β-carotene is commonly found in
all-E, as well as in its
9Z- and
13Z- isomers; lycopene also occurs as
5Z-,
9Z-,
13Z-,
15Z- and
di-Z-, while
9Z-lutein and
13Z-lutein have been identified in tomatoes
[9].
Figure 2. Examples of chemical structures of trans (All-E) and cis isomers (15Z-, 13Z- and 9Z-) of lycopene.
Chirality is another characteristic of most carotenoids due to the presence of chiral centers in their molecules. Astaxanthin and zeaxanthin are characteristic examples of chiral carotenoids, both with two chiral centers in 3 and 3′ carbon due to the presence of hydroxyl groups. These carotenoids have two enantiomers—the 3R,3′R and the 3S,3′S—and an optical inactive meso form of 3R,3′S
[11].
2. Sources of Natural Carotenoids
Carotenoids are naturally occurring in plants, photosynthetic bacteria and algae but also in some heterotrophic bacteria and fungi
[6][7]. Animals and humans obtain carotenoids from food, as they are unable to synthesize them. Nevertheless, the obtained carotenoids can be modified with metabolic reactions
[6] for the synthesis of other carotenoids or their derivatives.
2.1. Plants
Plants constitute the prevalent source of natural carotenoids, as a wide spectrum of these compounds is found in fruits, flowers and vegetables but also in other plant tissues such as leaves, roots and seeds
[12].
β-carotene is the predominant carotenoid extracted from plants, with carrots, spinach, tomatoes, sweet potatoes, broccoli and lettuce to be the richest sources. According to Stephen et al.
[13],
α- and
β-carotene are the signature carotenoids of carrot, lycopene is for tomatoes, watermelon and papaya, lutein is for watercress and spinach and yellow bell peppers are rich in violaxanthin.
The utilization of waste fruits and vegetables for the sustainable recovery of carotenoids has been already demonstrated
[14]. Another interesting approach has been recently introduced by Metličar et al.
[15][16], proposing the exploitation of invasive alien plant species such as Japanese knotweed and Bohemian knotweed to extract carotenoids and xanthophylls.
2.2. Microorganisms
Besides plants, several microbial species, belonging to algae, bacteria and fungi, are able to accumulate intracellularly different types of carotenoids as metabolic products. Carotenoids production in non-photosynthetic microorganisms is connected to an evolutionary response to photo-oxidative damage caused by light and oxygen-rich habitats
[17]. In the last few decades, many research groups have prioritized the investigation on the biotechnological production of carotenoids. It is only indicative that from the 750 naturally occurring carotenoids, more than 600 can derive from microorganisms
[18][19].
Plant-derived pigments display difficulties with respect to characterization and standardization due to the effect of cultivation and climate conditions. Another important drawback lies in the stability and functionality of these pigments, particularly regarding exposure at high temperatures, pH variations and light
[20]. Microorganisms evidence a prominent scientific potential due to the variation of their pigment color, the chemical profile of the pigment, the independence of seasonal restrictions and climate conditions, along with the capacity to scale up. In the framework of sustainability and circular economy, it is nowadays imperative to explore alternative sources of food additives that will not compete with food and feed and, in parallel, will respect the environment. Microbial carotenoids coincide entirely with the above mentioned purposes. The efficient productivities and high yields, complete process control and standardization of the product quality confer advantages of microbial carotenoids’ synthesis, directly related to the potential for large-scale production. Moreover, the utilization of low-cost and renewable resources (such as agro-industrial wastes and byproducts) as substrates for microbial growth could further mitigate the overall cost of production
[21].
3. Biotechnological Production of Carotenoids
Table 1 refers to indicative research of the last five years on the biotechnological production of carotenoids. In fact, algae and fungi are the most popular choices among the most studied microbial strains for carotenoid production such as β-carotene, astaxanthin, torulene, zeaxanthin, torularhodin and lutein.
Microalgae are widely recognized as sources of diversified bioactive compounds such as pigments, phenolic compounds, fatty acids, proteins and vitamins, among others
[17]. The microalgae
Haematococcus pluvialis and
Dunaliella salina have been extensively studied for astaxanthin and
β-carotene production, respectively
[17][22][23][24]. Spirulina is richer in
β-carotene even compared to carrots
[20].
Chlorella and
Scenedesmus are also significant carotenoid producers, mainly of lutein
[25][26]. The limitation of large-scale carotenoid production using microalgae lies in the high production costs and the high land requirements
[21][27]. Bacteria and fungi introduce a prevalent advantage to this angle, as proper strain selection, substrate and fermentation/bioreactor design can lead to high product yields and productivities in completely controlled processes.
Yeasts have emerged as robust carotenoid producers, with the strains
Rhodotorula sp. and
Phaffia rhodozyma occupying most of the recently published works.
Rhodotorula sp. together with the genera
Rhodosporidium,
Sporidiobolus and
Sporobolomyces belong to a category known as “red yeasts”, describing their ability to intracellularly accumulate carotenoids
[28]. Another important attribute of yeasts is their capability to synthesize carotenoids via the valorization of low-cost substrates as growth substrates, including cheese whey
[21], molasses
[29] and raw glycerol
[30]. Red yeasts have been generally reported to produce mixtures of carotenoids, mainly
β-carotene,
γ-carotene, lycopene, torulene and torularhodin
[21][28].
P. rhodozyma, a basidiomycetous yeast, is a well-known producer of astaxanthin and
β-carotene
[20][31]. The fungus
Blakeslea trispora is industrially employed for
β-carotene and lycopene production and its use as a food additive has already been approved
[32][33].
Likewise, recent studies have been undertaken using some Archaea for carotenoid synthesis. Many Haloarchaea have developed the ability to accumulate pigments as a response to stress factors. To this end, Giani et al.
[34] investigated the potential of the strain
Haloferax mediterranei to synthesize carotenoids, and especially the C
50 carotenoid bacterioruberin, when subjected to various concentrations of H
2O
2. Similarly Lizama et al.
[35] explored different Haloarchaea strains, namely
Halorubrum tebenquichense and
Haloarcula sp., for their carotenoid profile and antioxidant capacity.
Finally, cyanobacteria are also able to produce carotenoids, still in lower amounts compared to other pigments such as phycocyanin
[36]. Pagels et al.
[36] recently reviewed the potential of these microorganisms, reporting the strains
Cyanobium sp.,
Arthrospira platensis,
Trichodesmium sp. and
Lyngbya sp. as some characteristic examples of carotenoid-producing strains, also exhibiting antioxidant activities. The main carotenoids synthesized by these strains were
β-carotene and zeaxanthin.
Table 1. Recent developments on natural carotenoids produced by microorganisms.