Production of Carotenoids by Microorganisms

Abstract

Carotenoids are one of the most studied groups of biologically active compounds. They have antioxidant, anti-inflammatory, anti-cancer, and coloring properties and are particularly interesting for the pharmaceutical, nutraceutical, food, feed, cosmetic, and textile industries. Rapidly growing consumer demand for natural products has led to a significant increase in research and development of opportunities for their production from natural sources. Among the sources of carotenoids of natural origin, various microorganisms are of greater interest. This mini-review briefly summarizes the information published mainly during the last decade about carotenoid-producing microorganisms, the physiological importance of carotenoids for microbial cells, and the possibilities to improve their biosynthesis. This review also describes some new approaches/directions to make biotechnological production of microbial carotenoids more efficient.

1. Introduction

Carotenoids are one of the most studied groups of biologically active compounds. Information about their wide biodiversity, as well as about the factors affecting their composition and biological activity, has been very actively investigated and attracted increasing attention in the last decade [1,2,3,4,5,6].

They are lipophilic tetraterpenoid pigments produced by plants, algae as well as various microorganisms. Carotenoids are natural secondary metabolites that have antioxidant, anti-inflammatory, anti-cancer, and coloring properties. In this regard, they have a serious interest in the pharmaceutical, nutraceutical, food, feed, cosmetic, and textile industries. An increasing number of studies of the nature of carotenoids, their most effective producers and conditions for maximum synthesis, and the development of their practical application indicate a huge interest in this group of biologically active compounds.

The global market is still dominated by synthetic carotenoids. However, the rapidly growing consumer demand for natural products has led to a significant increase in research and development aimed at developing opportunities for their production from alternative natural sources. This applies to both the extraction of carotenoids from plant raw materials and their microbial synthesis (Figure 1). Among the sources of carotenoids of natural origin, the microalgae Haematococcus pluvialis, Dunaliella salina, Botryococcus braunii, Blakeslea trispora fungi, and various types of yeast and bacteria are of greater interest. The most practically important carotenoids include astaxanthin, beta-carotene, lutein, zeaxanthin, canthaxanthin, lycopene, and some other compounds [7]. Currently, the most well-known are beta-carotene (a precursor to vitamin A), necessary for eye health, as well as lycopene and astaxanthin, which can reduce the risks of cancer and atherosclerosis [8,9]. Increasing opportunities for the use of carotenoids in medicine are leading to a rapid increase in their market value. According to expert forecasts, by 2025, it will reach 1.7 billion US dollars [10].

Carotenoids are potent antioxidants. Thanks to this, on the one hand, they can protect the organisms synthesizing them from oxidative stress caused by various factors of the surrounding environment [11,12]. On the other hand, they are of great interest for the treatment of chronic degenerative, malignant, and viral diseases of humans [13] associated with oxidative stress.

Various microorganisms are of great interest as producers of carotenoids for their industrial biotechnological production. This is due to the possibilities of their controlled cultivation, minimization of the production period, and independence from the season. The efficiency of the production process depends on the adequate selection of the microbial strain. The undoubted advantage of microorganisms—potential producers of carotenoids is the high plasticity of their metabolism. It allows the use of relatively cheap media for their reproduction, including waste from crop production and various branches of the biotechnological industry. In recent years, increasing attention has been paid to the study of marine microorganisms and, in particular, marine archaea, in which a large variety of carotenoids is identified, including new ones [14,15,16]. It is believed that the use of marine microorganisms will make it possible to obtain a wide spectrum of carotenoids in a renewable way without depleting natural resources. Interest in bacterial pigments of marine origin due to this and the expected interesting potential for their use has been increasing rapidly recently [17]. The unusual marine environment is associated with great chemical diversity, which leads to the emergence of new biologically active compounds. Thus, marine organisms may represent an important source of novel biologically active compounds of significant interest for practical use in the future. However, it should be noted that the lack of production standards, clinical trials, and toxicity analysis still limits this research direction [18].

This mini-review briefly summarizes the information published mainly during the last decade about carotenoid-producing microorganisms, the physiological importance of carotenoids for microbial cells, and the possibilities to improve their biosynthesis. At the same time, the focus is on fungi and yeast and, to a lesser extent, bacteria and archaea. This review also describes some new approaches/directions to make biotechnological production of microbial carotenoids more efficient.

2. Physiological Role of Pigments in Microorganisms

Using many microorganisms widely distributed in nature, characterized by a huge variety of species, morphology, and pigmentation, it has been shown that the ability to synthesize a variety of pigments, including carotenoids, provides them with an advantage for survival in changing habitat conditions (Figure 2). The ability to synthesize pigments as an adaptive means of protecting against various damaging factors was formed during evolution under the influence of external environmental stresses, such as ultraviolet light, radiation, nutrient restriction, and increased osmotic pressure [12]. This idea is supported by the evidence that pigmented strains are more resistant to UV than non-pigmented strains [19,20]. In photosynthetic and non-photosynthetic bacteria, carotenoids, as auxiliary photosynthetic pigments, act as photoprotectors and antioxidants, thereby protecting cells from damage caused by UV radiation and sunlight [11]. Carotenoids are associated with several important biological functions, such as antenna pigments in photosynthesis and protection against oxidative stress. They are also involved in the microbial membrane’s adaptive response to low temperatures by regulating membrane fluidity. Moreover, they are linked with the increased resistance of organisms to freeze-thaw stress. The synthesis of carotenoids is transcriptionally activated under low-temperature conditions. Therefore, it can be considered an integral part of a controlled and regulated adaptive response to low-temperature conditions [21].

An assessment of the role of carotenoid pigments of bacteria isolated in the Antarctic showed their protective function against cryo- and solar radiation. Thus, a higher resistance of pigmented strains of microorganisms was demonstrated compared to unpigmented ones when subjected to stresses caused by 12-h freeze-thaw cycles, as well as artificial solar radiation (300 W/m²) [22].

The identification of heterotrophic bacterial pigments provides information about the physiological role they can play in the body. For example, Arthrobacter sp. NamB2 pink pigment, playing a role in this phenotype [23], consists of a mixture of carotenoid bacterioruberin and its dehydrated/glycosylated variants common in bacterioruberin-based pigment complements. This allowed the hypothesis that the pink carotenoid pigmentation of Arthrobacter sp. NamB2 plays a protective role in Namibia’s deserts and makes a significant contribution to its radiation resistance [24]. These assumptions are supported by the results of another earlier study that showed the role of bacterioruberin in the UV resistance of Haloarchaea [25].

The photoprotective role of the pigment was shown by comparing pigmented and naturally occurring unpigmented strains of Arthrobacter sp. NamB2. High carotenoid content in microbial cells during the stationary growth phase was found to improve their survival. The accumulation of torularhodine has been shown as an important mechanism to increase yeast resistance to UV-B [26]. In experiments with bacterial cells, it was shown that Microbacterium sp. LEMMJ01 isolated from the Antarctic soil had very high survival rates under UV radiation. Besides, the enrichment of E. coli K12A15 wild-type cells with the whole pigment extract from Microbacterium sp. LEMMJ01 led to the appearance of their resistance to UV radiation and was linked with the ability to repair DNA damage. The analyses of the crude pigment fraction revealed the existence of neurosporene, α-carotene, echinenone, canthaxanthin, and astaxanthin with a significant photoprotective effect [27]. It is supposed that the increased resistance of microorganisms to radiation and oxidation is linked also with carotenoids’ role in cellular membrane modifications [26]. As well as terrestrial phototrophic prokaryotes, a variety of heterotrophic prokaryotes of marine origin belonging to the types Bacteroidota (formerly known as Bacteroidetes) and Pseudomonadota (formerly known as Proteobacteria) accumulate carotenoids to protect against photooxidative damage and to regulate membrane fluidity [18]. It was also shown recently that E. coli cells treatment with carotenoids essentially decreased membrane damage caused by butanol. The results of this study demonstrate the potential biotechnological application of carotenoids to improve the economics of microbial butanol production [28].

At the same time, opportunistic Staphylococcus aureus regulates the lipid composition of membranes during adaptation to aggressive human body conditions by the synthesis of carotenoids that protect it against antimicrobial agents. These carotenoids, protecting S. aureus in this way, act as virulence factors for humans [29,30].

It is assumed that pigments of microorganisms, being secondary metabolites, play a key role that has not yet been fully identified, not only in protecting cells but also in environmental interactions with other organisms [31]. Studies of fungal mutants with altered pigmentation have shown how these pigments can provide a survival advantage to pathogenic fungi in the host organism, helping to evade its immune system [32].

Thus, microbial pigments play many roles in the fitness of various organisms for survival under adverse environmental factors and, therefore, in building ecosystems [33].

3. Carotenoid-Producing Microorganisms

At present, it remains relevant to search for various microorganisms-producers of carotenoids and to study the ways of their biosynthesis, including the obtaining of genetically improved strains. Production strategies are studied, as well as methods for extracting and purifying the desired product [34,35,36]. Combined efforts in this direction will make it economically feasible to produce carotenoids on a commercial scale.

3.1. Fungi

Different carotenogenic fungi can be isolated from various habitats, which are classified according to their ecological role: halophilic, acidophilic, psychrophilic, or thermophilic. A close relationship has been established between their tolerance towards the features of their habitats and the huge variety of pigments they synthesize [37,38].

Filamentous fungi, especially ascomycetes, produce secondary metabolites containing a wide range of pigments, including β-carotene, melanins, azaphilones, quinones, flavins, ankaflavin, monascin, anthraquinone, and naphthoquinone, giving different colors and shades. In addition to coloring properties, other useful properties of fungal pigments, such as antimicrobial, anticancer, antioxidant, antiproliferative, and cytotoxic activities, expand the scope of their practical use [39,40].

Compared to other groups of organisms, some types of fungi, including zygomycete, ascomycete, basidiomycete, and asexual deuteromycete species, produce higher levels of carotenoids comparable to carotenoid concentrations per unit biomass in microalgae. At the same time, given the close phylogenetic relationship of some fungi with humans, they can be considered excellent model systems for studying the role of carotenoids in human health and aging [41].

A common feature of fungal and other organisms’ carotenoids is their ability to inactivate oxygen radicals and singlet oxygen [42]. However, most carotenoids differ in their chemical structure.

Currently, industrial production of β-carotene and its precursor lycopene is based on the use of a heterothallic zygomycetous fungus, Blakeslea trispora. In B. trispora, β-carotene is biosynthesized through the mevalonate pathway. β-carotene of this fungi is the first permitted microbial food dye in the European Union [43]. The market value of carotenoids produced using B. trispora is expected to reach $2.0 billion in 2026 [44]. Industrial production of β-carotene is based on the cultivation of two type mating-type strains of B. trispora—plus (+) and minus (−) and their sexual interaction [45]. Various cheap substrates and industrial wastes may be used in carotenoid production [46,47,48]. It was shown that the optimization of the substrate based on the use of bug-damaged wheat gave the possibility to increase the amount of produced β-carotene approximately 3.5 times [47]. Another study showed that the addition of soy oil and cottonseed oil into fermentation media also increased the production of carotene [49]. Several studies published during the last decades were directed to the search for efficient stimulators of β-carotene production by B. trispora. There are non-ionic surfactants and natural oils (such as palm oil, sunflower oil, and soybean oil), arachidonic acid, and ethylene, as well as various oxidative stress inducers [50,51,52,53,54,55]. Interesting results of the research in this direction were published recently. Two factors were used in this study—bacteria Kocuria rhizophila as a microbial stimulant and butylated hydroxytoluene as an oxidative stress inducer. Used separately, they increased the production of carotenoids by 2.3 and 2.4 times, respectively, but when these factors were used jointly, there was a 7.5-fold increase in the number of synthesized carotenoids [56]. Another approach was proposed by Ge et al. (2022) [57]. They showed that the amount of β-carotene produced by B. trispora might be increased 3-fold when the culture was transferred from darkness to blue light for 24 h. It was revealed in this study that 1124 genes were upregulated and 740 genes were downregulated, respectively, after blue light exposure. This research, for the first time, provided gene expression differences between blue light and dark conditions at a whole genome level [57]. Despite the high potential for using B. trispora in the industrial production of β-carotene, the mechanism of light-induced carotenoid biosynthesis and the functioning of structural and regulatory genes in carotenogenesis is still unclear.

3.2. Yeast

Big attention in searching for the producers of carotenoids attracts a heterogeneous group of basidiomycetes described as red yeast. They are a source of highly effective microbial carotenoids and are easy and cheap to cultivate. The synthesized carotenoids are divided into two groups: carotenes containing only carbon and hydrogen atoms (C₄₀H_x) ‘oxygen-free carotenes’ e.g., α-carotene, β-carotene, γ-carotene, lycopene, and torulene and the second group that besides carbon and hydrogen contain oxygen atoms (‘oxygen-containing xanthophylls’) (C₄₀H_xO_y). This group includes astaxanthin, lutein, zeaxanthin, β-cryptoxanthin, fucoxanthin, and canthaxanthin [4,58,59,60].

Astaxanthin is of interest as a pigment exhibiting antimicrobial, particularly antifungal and anti-biofilm activity against Candida albicans and Candida glabrata [61]. Carotenoids synthesized by yeast of the genera Rhodotorula and Sporobolomyces also often show strong antimicrobial activity [62,63,64]. Of interest is the use of the carotenoid torularhodin produced by R. rubra as an antibacterial substance to solve the problem of attachment and reproduction of microorganisms on the surface of implants and medical devices. Its use in the composition of the coating provides its antimicrobial activity against some standard bacterial strains, such as Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, and Pseudomonas aeruginosa [65]. One more carotenoid producer between yeasts is Phaffia rhodozyma (also known as Xanthophyllomyces dendrorhous). It produces β-carotene and astaxanthin [9]. This organism is accepted as GRAS by the FDA (FDA 2000). The biochemical composition of this yeast is well controllable by varying the culture conditions. For the production of its biomass, it is possible to use inexpensive substrates, including various industrial and agricultural waste. Ph. rhodozyma yeast may be used as a commercial source of astaxanthin [66].

Due to the growing demand for natural carotenoids, the search for new yeast strains capable of efficiently synthesizing these compounds remains relevant. In the studies of scientists from Poland, 114 strains of red yeasts were isolated from various natural media and food samples [67], and a lot of them synthesized a large amount of torularhodin. The strains identified belonged to six genera: Rhodotorula, Sporidiobolus, Sporobolomyces, Buckleyzyma, Cystofilobasidium, and Erythrobasidium. The largest number of isolates belonged to Rhodotorula babjevae (18), Rhodotorula mucilaginosa (7), Sporidiobolus pararoseus (4), and Rhodotorula glutinis (4). Several species of yeast and bacteria from the genera Rhodotorula, Sporobolomyces, Sporidiobolus, Gordonia, and Dietzia could potentially become sources of carotenoids on an industrial scale, but the technological solutions available in this direction still need to be improved [7]. Rhodotorula yeast is of considerable interest as a valuable source of metabolites with various natural biological activities, the main ones being carotenoids. Information on the current state of microbial biotechnology using Rhodotorula yeast species, an analysis of their production efficiency with some information on the subsequent stages of extraction of biomolecules with high added value, as well as information on their potential use, is presented in the recent review of Mussagy et al. (2022) [68]. This review also discusses new genetic engineering technologies, indicating some areas of their possible use. Biotechnological interest may include yeast R. mucilaginosa, which is capable of growing on a wide range of substrates and has high-stress resistance. This yeast is an excellent producer of carotenoids, lipids, enzymes, and other functional products of interest as potential alternative sources of food components beneficial to public health. These compounds are also biodiesel production substrates, dyes, and functional ingredients for cosmetics. The biosynthetic pathways of carotenoids, lipids, and enzymes, as well as the effect of specific fermentation factors on their productivity, have been studied in this yeast [69]. The main carotenoids synthesized by R. mucilaginosa are β-carotene, torulene, and tolarhodine [70], which are antioxidants, vitamin A precursors, cancer inhibitors, and immune activators [59]. The glucoside carotenoid extracted from the yeast R. mucilaginosa AY-01 showed significant antagonistic activity against antibiotic-resistant bacteria [71]. It has been demonstrated that the osmophilic strain R. toruloides C23 has excellent carotenogenic ability and good growth when using different carbon sources. Comparative transcriptomic analysis showed that this strain developed a special molecular regulation mechanism to maintain a high simultaneous accumulation of intracellular carotenoids and cell growth at high sugar concentrations. This allows the yeast R. toruloides to be considered as an alternative source of carotenoids consisting of β-carotene, torulene, and torularhodine [72,73,74]. A recent review provides comprehensive information on carotenoid biosynthesis studies in R. toruloides, focusing on the identification of biosynthetic pathways and the regulation of key enzymes and genes involved in this process [75].

Often, red yeast produces a mixture of carotenoids, and when it is necessary to obtain a particular pigment, expensive multi-stage purification is required. In this regard, several studies have been aimed at developing approaches that increase the synthesis of specific carotenoids by these yeasts, such as, for example, β-carotene [76]. Genetic engineering and molecular biology tools are used to increase the synthesis of the necessary carotenoids, aimed at controlling gene expression in metabolic pathways involved in the synthesis of carotenoids [77]. A review by Akaraphol Watcharawipas and Weerawat Runguphan (2023) [58] shows how random mutagenesis and rational metabolic engineering mediated by synthetic biology tools could be powerful strategies to improve carotenoid production in red yeast. Understanding how they synthesize carotenoids and finding ways to increase their synthesis using inexpensive substrates is critical to achieving an economically competitive process for the production of valuable carotenoids.

To date, research has focused mainly on the synthesis of C40 carotenoids. However, advances in metabolic engineering have contributed to the discovery of novel C30-carotenoid compounds with greater antioxidant activity. This contributed to increased interest in developing a strategy for their optimized synthesis [78].

3.3. Bacteria

In recent years, carotenoids from bacteria have been of great interest since there are suggestions that carotenoids of prokaryotic origin may have higher efficacy. Bacteria may be preferred also because of their genetic simplicity. The advantages of bacterial pigments over synthetic pigments, their production strategies, and the importance of bacterial pigments in various industries are well described by Priya Sundararajan and Shanmuga Priya Ramasamy (2023) [79]. Some bacteria can synthesize both C40-carotenoids and C30-carotenoids. Both terrestrial phototrophic prokaryotes and a variety of marine heterotrophic prokaryotes belonging to the Bacteroidota (formerly known as Bacteroidetes) and Pseudomonadota (formerly known as Proteobacteria) types have been found to accumulate carotenoids for protection against photooxidative damage and control of membrane fluidity [18]. The biosynthesis of carotenoids in them can also be considered as an adaptive response to certain adverse effects of the marine environment, such as excessive solar radiation and reactive oxygen species. The advantage of using marine organisms as a source of carotenoids over chemical synthesis is their higher activity, bioavailability, and stability. In addition, marine organisms synthesize a wide variety of carotenoids that can be obtained without depleting natural resources. A study of microbial biodiversity in tidal flats has identified a new species, Croceibacterium, belonging to the family Erythrobacteraceae, accumulating predominantly zeaxanthin [80]. Endosymbiotic bacteria can become a source of unique compounds with powerful biomedical applications. Thus, pigmented endophytic bacteria selected from Avicennia marina mangrove explants and identified as Micrococcus luteus produce an intracellular yellow pigment exhibiting significant dose-dependent antioxidant and anticancer activity [81]. Significant antibacterial activity of halophilic bacteria carotenoids was also noted in other works [82,83].

The recent paper described the results of a screening and characterization of pigment profile and photostability in seventy-four newly isolated Antarctic bacteria using Fourier-transform (FT) Raman spectroscopy and HPLC-MS. Thus, several bacteria with high pigment content were isolated from the genera Agrococcus, Arthrobacter, Brachybacterium, Cryobacterium, Leifsonia, Micrococcus, Paeniglutamicibacter, Rhodococcus, Salinibacterium, and Flavobacterium. HPLC-MS showed the presence of 18 different carotenoids and precursors in 10 pigmented Antarctic bacteria. Moreover, blue light increased pigment production in most bacteria, while the effect of temperature was strain-specific. This study provides valuable insights into the pigment production capabilities of Antarctic bacteria [84].

3.4. Archaea

Archaea are of great interest for the discovery of new metabolites. This is due to their ability to adapt to extreme environmental conditions and their specific metabolic pathways leading to the synthesis of unique biomolecules. Studies of biotechnological and pharmacological properties, as well as the possible industrial production of archaeal carotenoids, are relatively few. Current data on carotenoid metabolism in archaea, their classification and biological properties, as well as the potential use of these pigments in biotechnology and medicine, are presented in a review by Grivard et al. (2022) [85]. Archaea are presented as promising microorganisms for the production of carotenoids on a large and medium scale since fast and cheap culture systems can be combined with simple subsequent extraction and purification processes. Carotenoids produced by halophilic archaea may play a dual role—membrane stabilizers and active antioxidants. Halophilic archaea are known to produce 25 different carotenoids, 13 of which are highly specific [86]. Carotenoids of Haloferax sp. ME16, Halogeometricum sp. ME3 и Haloarcula sp. BT9 isolated from the salt lakes of Algeria demonstrated the heterogeneity of their composition depending on the strain with a predominance of bacterioruberin. Assessment of their antioxidant capacity showed that it exceeded the corresponding indicators of ascorbic acid used as a standard. The antibacterial activity of carotenoids against four strains pathogenic for humans and four strains pathogenic for fish, assessed by the agar disk diffusion method, was quite high, and this suggests that the C50-carotenoids of the studied strains offer promising prospects for their biotechnological use [87].

4. Effect of Culture Conditions on the Accumulation of Carotenoids by Microorganisms

Cells of microorganisms in nature are constantly exposed to changes in environmental conditions that determine their growth, metabolic activity, and survival. One of the protective reactions against various stresses is the synthesis of some specific metabolites, including carotenoids, by microorganisms. Taking into account the protective role of carotenoids accumulating in the cells of microorganisms under various extreme treatments, biotechnologists are faced with the possibility of both genetic and phenotypic effects on the processes of carotenogenesis. One strategy used to minimize the high costs of producing carotenoids by microorganisms is to optimize culture conditions to increase their yield. Some works consider the use of inexpensive and affordable substrates, the selection of process conditions, such as the use of various sources of carbon and nitrogen, their ratio, stimulation of the synthesis of carotenoids with various additives, including trace elements, intermediates, NaCl, H₂O₂, light irradiation, as well as manipulations with pH, cultivation temperature, aeration, and other effects [7,8].

Carotenoids are known to be associated with important biological functions in photosynthesis as well as in protection against oxidative stress. As was shown above, they participate in the adaptive response of the membrane of the microorganism to low temperature, leading to increased resistance to stress during freezing-thawing. The synthesis of carotenoids is transcriptionally activated under low-temperature conditions; that is, it can be considered an integral part of a controlled and regulated adaptive response to low-temperature conditions [21]. A study of growth characteristics, biomass production, and carotenoids in the presence of exogenous stressors (NaCl, H₂O₂) in R. glutinis yeast showed that even when low concentrations of salt or peroxide were added to the medium, the synthesis of carotenoids increased by about two times, with a slight change in biomass. Greater stress caused a greater increase in the synthesis of carotenoids, while combined stress showed the highest results [88]. In Sporobolomyces pararoseus, considered an alternative source of carotenoids consisting of β-carotene, torulene, and torularodine, the carotenoid content increased significantly after 120 h of incubation with H₂O₂: after incubation with 40 mM H₂O₂, more than 2.3 times. The main carotenoids that increased during H₂O₂ treatment were β-carotene and torularhodin. The findings not only offer an affordable and effective way to stimulate carotenoid synthesis in S. pararoseus but also provide a molecular basis to further increase their production through genetic or metabolic engineering [74]. Oxidative, osmotic, and salt stress in other yeasts also contributed to the production of significantly more carotenoids [88,89]. The low temperature caused increased carotenoid biosynthesis in R. toruloides in environments containing agro-industrial waste—potato effluent and glycerol. The mechanism by which yeast can adapt to this stress is currently being investigated [90]. Induction of osmotic stress and low temperature intensified the biosynthesis of β-carotene in yeast organisms (up to 73.9% of the total carotenoid content). Under oxidative stress, yeast synthesized torulene more efficiently (up to 82.2%) than under other conditions, while white light irradiation increased torularhodin production (up to 20.0%) [91]. Cultivation of R. toruloides yeast under osmotic stress stimulated carotenoid production by 10% [92]. R. toruloides have also been used in the investigation of the response to light involved in carotenoid biosynthesis. Results from phenotype and gene expression analysis showed that in R. toruloides, light caused darker pigmentation and an associated increase in carotenoid production [93]. The high temperature increased the yield of torulene and torularhodin, which may be due to an increase in the production of acetyl-CoA formed as a result of activation of the TCA cycle pathway, as well as increased regulation of pathways directly related to carotenogenesis. These studies show the possibility of intensifying the fermentation process to improve the production of carotenoids and provide a molecular basis for further increasing their yield by genetic engineering [94]. The production of carotenoids in haloarchea can also be improved by optimizing the culture medium in terms of salinity, pH, and temperature [83]. It has been shown that oxidative stress induced by hydroperoxides and reactive oxygen species in the production of carotenoids on a medium of used cooking oil and corn extract by B. trispora fungus causes a significant increase in their production. The resulting carotenoids consisted of β-carotene (71%), γ-carotene (26%), and lycopene (3%) [54].

Fairly inexpensive production of both red yeast and other carotenoid producers, including fungus B. trispora, can be based on the use of various waste products from the processing industry and raw agricultural materials. For their cultivation, it is possible to use substrates such as beet molasses, cheese whey, raw vegetable oils, crude glycerol, cabbage waste, watermelon husks, peach peel, waste cooking oils, as well as wastewater formed during the processing of green olives, producing not only valuable carotenoids but also reducing environmental pollution and reducing production costs [45,48,54,95,96,97,98]. The yield of carotenoids can be improved by optimizing the medium and modifying the extraction methods [99].

One promising low-cost carbon source is crude glycerol, a byproduct of biodiesel production. According to Saenge et al. (2011) [100], the R. glutinis strain, when grown on crude glycerol, reached the maximum carotenoid production by adding the surfactant Tween 80 to the culture medium. Metabolomic analysis of R. toruloides cultured on glycerol as the sole carbon source showed that the tricarboxylic acid cycle and amino acid biosynthesis are suppressed, and metabolic fluxes are shifted towards lipid and carotenoid synthesis [101].

In a study of the production of carotenoids and lipids by yeast, R. toruloides cultivated on wheat straw hydrolysate, β-carotene, γ-carotene, torularhodin, and torulene were identified as the main carotenoids. The growth kinetics study showed a positive correlation between carotenoid content and lipid accumulation. β-carotene was the main carotenoid [102].

One of the problems of cultivation of carotene synthesizing yeast on lignocellulose-containing biomass is the insufficient tolerance of yeast cells to inhibitors formed during substrate pretreatment. Using R. toruloides yeast, an adaptive laboratory evolution strategy in a hydrolysate-based medium was developed to improve host strain tolerance. The evolved strains showed better growth characteristics in the hydrolysate-based medium with a significant reduction in lag phases and improved ability to produce carotenoids compared to the original wild-type strain. In the best cases, the lag phase was reduced by 72 h [103]. In a study of red yeast R. babjevae, a cost-effective fermentation process was developed for the production of carotenoids. For this, lignocellulose wastes were used as raw materials. A method of detoxifying an acidic wheat bran hydrolysate has been proposed, which results in a reduction in the presence of inhibitors. Identification of produced carotenoids revealed the presence of torulene (52%), torularhodine (36%), and β-carotene (9.0%) [104]. When growing R. glutinis in olive plant effluents, the total yield of carotenoids increased significantly at low initial pH, high temperature, illumination, and the addition of certain amounts of urea and glycerol. It has been shown that by varying these conditions, it is possible to selectively influence the qualitative composition of the synthesized carotenoids. Thus, the yield of torularhodine can be increased by using high pH and low temperature and by adding urea and glycerol. To selectively induce the synthesis of torulene, cultivation should be carried out at low pH, high temperature, and illumination. In addition, low pH, high temperature, and addition of urea contributed to the high production of β-carotene. Up to 85.40, 80.67, and 39.45% of torulene, torularhodine, and beta-carotene, respectively, were obtained under the selected conditions [105]. For the production of carotenoids using R. glutinis, the use of an optimized medium based on goat milk whey [106] is also promising. In the study of torularhodine synthesized by the yeast Rhodotorula sp. when cultured in an optimized medium containing sorbitol and beef extract at pH 6.25, it was found its antimicrobial activities against E. coli and S. aureus. This allows its use as a natural antibacterial preservative in various foods [64].

Positive results on increasing the synthesis of lycopene by B. trispora culture were achieved with the addition of geraniol to the medium, as well as in experiments with the addition of isopentenyl alcohol, mevalonic acid, or dimethyl allyl alcohol [107]. When growing B. trispora on deproteinated hydrolyzed whey, the concentration of β-carotene was significantly influenced by the intensity of aeration. High productivity of β-carotene by B. trispora culture was detected when Tween 80, Span 80, and β-ionone were added to the medium [95]. It was shown that waste products enriched with carotenogenesis-stimulating substances increased both the content of β-carotene and the specific rate of its production. The use of hydroxytyrosol and tyrosol [48] contributed to the highest stability in the production process of β-carotene by B. trispora grown in enriched wastewater. In turn, sodium acetate as an additive for the production of β-carotene by B. trispora led to its increase since it induced the expression of five carotenogenesis genes [108].

5. Future Directions

Thus, carotenoids, essential for maintaining the life and well-being of humans, animals, and plants and which do not exhibit toxic effects, are compounds in great demand in the pharmaceutical, food, and cosmetic markets. While chemical synthesis of carotenoids has several disadvantages, including a complex extraction process, high cost and low yield of the final product, as well as harmful environmental impacts and safety issues caused by the detrimental health effects of synthetic molecules and resistance to antimicrobial drugs, the production of carotenoids by microorganisms is characterized by its speed, simplicity, and low cost, as well as the possibility of improvement by changing some aspects of the cultivation. The ability of carotenoids to exhibit immune-boosting effects by increasing or decreasing the activity of genes associated with antioxidants, anticancer, lipid-lowering, and immunomodulatory properties justifies broad prospects for the development of new methods for their use in many areas where the use of chemicals, including antibiotics, is undesirable.

This justifies further expansion of interest in the variability of pigment production, cultivation conditions, antimicrobial activity, extraction methods, and prospects for industrial scaling [109]. In recent years, it has been shown that diets enriched with carotenoids exhibit a wide range of anticancer effects, modulate apoptosis and blockade of metastasis, protect dopaminergic neurons of the brain from oxidative damage, and reduce the severity of neurodegenerative disorders, Alzheimer’s, and Parkinson’s diseases, correct metabolic disorders of diabetes mellitus and abdominal obesity, and reduce the risk of cardiovascular diseases. Since carotenoids have limited bioavailability due to their low water solubility, future research can be aimed at developing dosage forms of carotene-containing herbal preparations free of this drawback [110]. Of particular interest in subsequent studies may be Deinococcus bacteria, which are highly resistant under a variety of extreme environmental conditions, including dehydration, high temperatures, and ionizing radiation. It is believed that such resistance is largely due to their ability to synthesize carotenoids. It is assumed that they will have wide prospects for use in space exploration, where they can serve as radiation indicators and natural antioxidants to protect the health of astronauts during long-term space flights [111,112] and also be used to repair DNA damage caused by ionizing radiation [113].

6. Conclusions

Carotenoids are natural pigments with a variety of bright shades and have very useful properties. They are successfully used in the production of food and feed, pharmaceuticals, and cosmetics, as well as in the textile industry (Figure 3).

As was already mentioned in this review, increasing opportunities for the use of carotenoids lead to a rapid increase in their market value, which, according to expert forecasts, may reach 1.7 billion US dollars in 2025. Consumer interest in natural carotenoids, which are environmentally friendly and non-toxic, is largely due to the growing rejection of synthetic additives for food and personal hygiene products. All this, combined with low cost, ease, and speed of scaling, led to a sharp increase in market demand for naturally occurring carotenoids. Microbial carotenoids are particularly preferred because of their higher yield, stability, and cost-effectiveness. The great practical value of carotenoids, the possibility of their wide commercial use, and the economic efficiency of production contributed to the growing interest in the search for new and stable microbial producers, as well as the development of new cost-effective strategies for their production.

There are many different carotenoids, but only a few of them are produced on an industrial scale, including carotenes (β-carotene and lycopene) and xanthophylls (astaxanthin, lutein, zeaxanthin, canthaxanthin). Some biotechnological processes for the production of carotenoids were created several years ago. However, the search for new effective strains of producer microorganisms and the development of more advanced technologies for widely demanded carotenoid preparations continues. They also include proposals to use various agricultural and industrial wastes for the cultivation of the strains-producers. There are numerous directions in the further studies of this group of physiologically active substances, which allows us to hope for further success in the field of their successful use in human practice.