Carotenoid Production Research Articles

Simultaneous Lipid and Carotenoid Production via Rhodotorula paludigena CM33 Using Crude Glycerol as the Main Substrate: Pilot-Scale Experiments.

Rhodotorula paludigena CM33 is an oleaginous yeast that has been demonstrated to accumulate substantial quantities of intracellular lipids and carotenoids. In this study, crude glycerol, a by-product of biodiesel production, was used as a carbon source to enhance the accumulation of lipids and carotenoids in the cells. The culture conditions were first optimized using response surface methodology, which revealed that the carotenoid concentration and lipid content improved when the concentration of crude glycerol was 40 g/L. Different fermentation conditions were also investigated: batch, repeated-batch, and fed-batch conditions in a 500 L fermenter. For fed-batch fermentation, the maximum concentrations of biomass, lipids, and carotenoids obtained were 46.32 g/L, 37.65%, and 713.80 mg/L, respectively. A chemical-free carotenoid extraction method was also optimized using high-pressure homogenization and a microfluidizer device. The carotenoids were found to be mostly beta-carotene, which was confirmed by HPLC (high pressure liquid chromatography), LC-MS (liquid chromatography-mass spectrometry), and NMR (nuclear magnetic resonance). The results of this study indicate that crude glycerol can be used as a substrate to produce carotenoids, resulting in enhanced value of this biodiesel by-product.

Advances in the Discovery and Engineering of Gene Targets for Carotenoid Biosynthesis in Recombinant Strains.

Carotenoids are naturally occurring pigments that are abundant in the natural world. Due to their excellent antioxidant attributes, carotenoids are widely utilized in various industries, including the food, pharmaceutical, cosmetic industries, and others. Plants, algae, and microorganisms are presently the main sources for acquiring natural carotenoids. However, due to the swift progress in metabolic engineering and synthetic biology, along with the continuous and thorough investigation of carotenoid biosynthetic pathways, recombinant strains have emerged as promising candidates to produce carotenoids. The identification and manipulation of gene targets that influence the accumulation of the desired products is a crucial challenge in the construction and metabolic regulation of recombinant strains. In this review, we provide an overview of the carotenoid biosynthetic pathway, followed by a summary of the methodologies employed in the discovery of gene targets associated with carotenoid production. Furthermore, we focus on discussing the gene targets that have shown potential to enhance carotenoid production. To facilitate future research, we categorize these gene targets based on their capacity to attain elevated levels of carotenoid production.

Production of carotenoids from aromatics and pretreated lignocellulosic biomass by Novosphingobium aromaticivorans.

There is economic and environmental interest in generating commodity chemicals from renewable resources, such as lignocellulosic biomass, that can substitute for chemicals derived from fossil fuels. The bacterium Novosphingobium aromaticivorans is a promising microbial platform for producing commodity chemicals from lignocellulosic biomass because it can produce these from compounds in pretreated lignocellulosic biomass, which many industrial microbial catalysts cannot metabolize. Here, we show that N. aromaticivorans can be engineered to produce several valuable carotenoids. We also show that engineered N. aromaticivorans strains can produce these lipophilic chemicals concurrently with the extracellular commodity chemical 2-pyrone-4,6-dicarboxylic acid when grown in a complex liquor obtained from alkaline pretreated lignocellulosic biomass. Concurrent microbial production of valuable intra- and extracellular products can increase the economic value generated from the conversion of lignocellulosic biomass-derived compounds into commodity chemicals and facilitate the separation of water- and membrane-soluble products.

Building a machine-learning model to predict optimal mevalonate pathway gene expression levels for efficient production of a carotenoid in yeast.

Simultaneous modification of the expression levels of many metabolic enzyme genes results in diverse expression ratios of these genes; however, the relationship between gene expression levels and chemical productivity remains unclear. However, clarification of this relationship is expected to improve the productivity of useful chemicals. Supervised machine learning is considered to be an effective means to clarify this relationship. In this study, to improve the productivity of carotenoids in yeast Saccharomyces cerevisiae, we aimed to build a machine-learning model that can predict the optimal gene expression level for carotenoid production. First, we obtained data on the expression levels of mevalonate pathway enzyme genes and carotenoid production. Then, based on these data, we built a machine-learning model to predict carotenoid productivity based on gene expression levels. The prediction accuracy of 0.6292 (coefficient of determination) was achieved using the test data. The maximum predicted carotenoid productivity was 4.3 times higher in the engineered strain than in the parental strain, suggesting that the expression levels of the mevalonate pathway enzyme genes tHMG1 and ERG8 have a particularly large impact on carotenoid productivity. This study could be one of the important achievements in addressing the uncertainty of genotype-phenotype correlations, which is one of the challenges facing metabolic engineering strategies.

All Kinds of Sunny Colors Synthesized from Methane: Genome-Encoded Carotenoid Production by Methylomonas Species.

Carotenoids are secondary metabolites that exhibit antioxidant properties and are characterized by a striking range of colorations from red to yellow. These natural pigments are synthesized by a wide range of eukaryotic and prokaryotic organisms. Among the latter, carotenoid-producing methanotrophic bacteria, which display fast growth on methane or natural gas, are of particular interest as potential producers of a feed protein enriched with carotenoids. Until recently, Methylomonas strain 16a and Methylomonas sp. ZR1 remained the only representatives of the genus for which detailed carotenoid profile was determined. In this study, we analyzed the genome sequences of five strains of Methylomonas species whose pigmentation varied from white and yellow to orange and red, and identified carotenoids produced by these bacteria. Carotenoids synthesized using four pigmented strains included C30 fraction, primarily composed of 4,4'-diaplycopene-4,4'-dioic acid and 4,4'-diaplycopenoic acid, as well as C40 fraction with the major compound represented by 1,1'-dihydroxy-3,4-didehydrolycopene. The genomes of studied Methylomonas strains varied in size between 4.59 and 5.45 Mb and contained 4201-4735 protein-coding genes. These genomes and 35 reference Methylomonas genomes available in the GenBank were examined for the presence of genes encoding carotenoid biosynthesis. Genomes of all pigmented Methylomonas strains harbored genes necessary for the synthesis of 4,4'-diaplycopene-4,4'-dioic acid. Non-pigmented "Methylomonas montana" MW1T lacked the crtN gene required for carotenoid production. Nearly all strains possessed phytoene desaturases, which explained their ability to naturally synthesize lycopene. Thus, members of the genus Methylomonas can potentially be considered as producers of C30 and C40 carotenoids from methane.

Microalgae cultivation and carotenoid production in paint booth effluent mixed with domestic sewage

Microalgae cultivation and carotenoid production in paint booth effluent mixed with domestic sewage

Overproduction of Xanthophyll Pigment in Flavobacterium sp. JSWR-1 under Optimized Culture Conditions.

Flavobacterium can synthesize xanthophyll, particularly the pigment zeaxanthin, which has significant economic value in nutrition and pharmaceuticals. Recently, the use of carotenoid biosynthesis by bacteria and yeast fermentation technology has shown to be very efficient and offers significant advantages in large-scale production, cost-effectiveness, and safety. In the present study, JSWR-1 strain capable of producing xanthophyll pigment was isolated from a freshwater reservoir in Wanju-gun, Republic of Korea. Based on the morphological, physiological, and molecular characteristics, JSWR-1 classified as belonging to the Flavobacterium species. The bacterium is strictly aerobic, Gram-negative, rod-shaped, and psychrophilic. The completed genome sequence of the strain Flavobacterium sp. JSWR-1 is predicted to be a single circular 3,425,829-bp chromosome with a G+C content of 35.2% and 2,941 protein-coding genes. The optimization of carotenoid production was achieved by small-scale cultivation, resulting in zeaxanthin being identified as the predominant carotenoid pigment. The enhancement of zeaxanthin biosynthesis by applying different light-irradiation, variations in pH and temperature, and adding carbon and nitrogen supplies to the growth medium. A significant increase in intracellular zeaxanthin concentrations was also recorded during fed-batch fermentation achieving a maximum of 16.69 ± 0.71 mg/l, corresponding to a product yield of 4.05 ± 0.15 mg zeaxanthin per gram cell dry weight. Batch and fed-batch culture extracts exhibit significant antioxidant activity. The results demonstrated that the JSWR-1 strain can potentially serve as a source for zeaxanthin biosynthesis.

Effect of seawater on the biomass composition of Spirulina produced at a pilot-scale

The microalga Arthrospira platensis BEA 005B was produced in 11.4 m3 raceway photobioreactors and a culture medium based on commercial fertilisers and either freshwater or seawater. The biomass productivity of the reactors operated at a fixed dilution rate of 0.3 day−1 decreased from 22.9 g·m−2·day−1 when operated using freshwater to 16.3 g·m−2·day−1 when the biomass was produced using seawater. The protein content of the biomass produced in seawater was lower; however, the content of essential amino acids including valine, leucine and isoleucine was higher. Seawater also triggered the production of carotenoids and altered the synthesis and accumulation of fatty acids. For example, the biomass produced using seawater showed a 319% and 210% higher content of oleic and eicosenoic acid, respectively. The results demonstrate that it is possible to produce the selected microalga using seawater after an adaptation period and that the composition of the produced biomass is suitable for food applications.

Functional potential and evolutionary response to long-term heat selection of bacterial associates of coral photosymbionts.

Symbiotic microorganisms are crucial for the survival of corals and their resistance to coral bleaching in the face of climate change. However, the impact of microbe-microbe interactions on coral functioning is mostly unknown but could be essential factors for coral adaption to future climates. Here, we investigated interactions between cultured dinoflagellates of the Symbiodiniaceae family, essential photosymbionts of corals, and associated bacteria. By assessing the genomic potential of 49 bacteria, we found that they are likely beneficial for Symbiodiniaceae, through the production of B vitamins and antioxidants. Additionally, bacterial genes involved in host-symbiont interactions, such as secretion systems, accumulated mutations following long-term exposure to heat, suggesting symbiotic interactions may change under climate change. This highlights the importance of microbe-microbe interactions in coral functioning.

Effects of leachate concentration, carbon dioxide and aeration flow rate on chlorophyll and carotenoid productivity and bioremediation potential of the microalga Chlorella minutissima

The use of microalgae cultures to process effluents from industries, leachates, and tanneries, among others, quantified by the reduction of metallic materials in the medium and the reduction of chemical oxygen demand (COD), helps reduce the environmental impact caused by human development. In addition, with the growth of the culture, it is possible to produce a significant amount of chlorophyll, a carotenoid of high value in the cosmetics and food industries that are used as a natural pigment. In this context, this work presents a study conducted to verify the bioremediation and chlorophyll production potential of the cultivation of the microalgae Chlorella minutíssima, using the Taguchi method. The microalgae Chlorella minutissima has given good results in the bioremediation of leachate, as a mean reduction of 33% in COD was obtained, as well as a 92% reduction in the toxic components. In addition, statistical analysis revealed that the four process factors were significant factors for chlorophyll a, chlorophyll b and carotenoid productivity (p < 0.05). Finally, it was observed that the maximum chlorophyll a (111.9 ± 0.8 mg‧L−1‧d−1), chlorophyll b (66.1 ± 1.7 mg‧L−1‧d−1), and carotenoid (31.9 ± 0.03 mg‧L−1‧d−1) values obtained occurred in Experiment 8, which is closer to the ideal conditions identified by statistical analysis, revealing the effectiveness of the use of the Taguchi method for the design of experiments.

Carotenoids: Dietary sources, health functions, biofortification, marketing trend and affecting factors – A review

Nutritional deficiency is becoming a universal problem, resulting in poor health, low worker productivity, high rates of mortality and morbidity. Large-scale dietary improvements are needed to deliver a healthy, and sustainable diet to the increasing world population. Fruits and vegetables as well as their valorized wastes and industrial by-products constitute the major dietary sources of bioactive compounds, including carotenoids. Carotenoids are lipophilic pigments that occur widely in nature and are distributed in plants, fish, birds, algae, archaea, yeasts, fungi, bacteria, and invertebrate animals (such as insects, mites, aphids). The most significant aspects of carotenoids in the human diet are the antioxidant and provitamin A activities, and also the color that they impart to our food. The most researched carotenoids in diets and commonly occurring in the blood are β-carotene, β-cryptoxanthin, lycopene, lutein and α-carotene, with β-carotene comprising the major portion of pro-vitamin A. Phytoene and phytofluene are also recently becoming increasingly topical in the human diet. Genotype, environment, climate, postharvest handling and processing are among the major factors affecting carotenoids. Thus, in this review, results from several studies on recent developments of carotenoid dietary sources, marketability, biofortification, new insights for the sustainable valorization of food wastes and agro-industrial by-products, human health effects and factors affecting carotenoid concentrations and compositions are critically pinpointed and briefly discussed. Lastly, research needs, mainly on crop biofortification, waste valorization, therapeutic properties are highlighted so as to enhance carotenoid production and nutritional values with potentially significant health benefits.

Plant mediated fabrication of silver nanoparticles, process optimization, and impact on tomato plant

Nanotechnology is one of the fastest-growing markets, but developing eco-friendly products, their maximum production, stability, and higher yield is a challenge. In this study, silver nanoparticles were synthesized using an easily available resource, leaves extract of the Neem (Azadirachta indica) plant, as a reducing and capping agent, determined their effect on germination and growth of tomato plants. The maximum production of silver nanoparticles was noted at 70 °C after 3 h of reaction time while treating the 10 ml leaf extract of Neem plant with 10 ml of 1 mM silver nitrate. The impact of the extract preparation method and solvent type on the plant mediated fabrication of silver nanoparticles was also investigated. The UV-spectrophotometric analysis confirmed the synthesis of silver nanoparticles and showed an absorption spectrum within Δ420–440 nm range. The size of the fabricated silver nanoparticles was 22–30 nm. The functional groups such as ethylene, amide, carbonyl, methoxy, alcohol, and phenol attached to stabilize the nanoparticles were observed using the FTIR technique. SEM, EDX, and XRD analyses were performed to study the physiochemical characteristics of synthesized nanoparticles. Silver nanoparticles increased the germination rate of tomato seeds up to 70% while decreasing the mean germination time compared to the control. Silver nanoparticles applied at varying concentrations significantly increased the shoot length (25 to 80%), root length (10 to 60%), and fresh biomass (10 to 80%) biomass of the tomato plant. The production of total chlorophyll, carotenoid, flavonoids, soluble sugar, and protein was significantly increased in tomato plants treated with 5 and 10 ppm silver nanoparticles compared to the control. Green synthesized silver nanoparticles are cost-effective and nontoxic and can be applied in agriculture, biomedical, and other fields.

Sustainable approach to recover β-carotene and astaxanthin from Phaffia rhodozyma grown in a stirred-tank bioreactor under the influence of magnetic fields

This study aimed to produce carotenoids by Phaffia rhodozyma in a stirred-tank bioreactor under the influence of magnetic fields (MF) and to evaluate a sustainable approach to recover them from the yeast biomass. MF application proved to be effective in increasing 8.6 and 22.9 % of β-carotene and astaxanthin production, respectively. Regarding solid–liquid extraction (SLE), the ability of aqueous and ethanolic solutions of protic ionic liquids (PILs) was determined. β-carotene and astaxanthin recovery yields increased with the anion alkyl chain length hydrophobicity. [Pro][Oct]:EtOH (50 % v v-1) was selected as the effective solvent. Moreover, it led to improvement in carotenoid stability at different storage temperatures over time in comparison with the control. This study is one of the first to describe an effective and sustainable approach to move carotenoid production from shake flasks to a bioreactor under the influence of MF and recover carotenoids from P. rhodozyma biomass.

The sensor histidine kinase (SLN1) and acetyl-CoA carboxylase (ACC1) coordinately regulate the response of Neurospora crassa to the springtail Sinella curviseta (Collembola: Entomobryidae) attack.

Understanding the regulatory pathways by which fungi respond to environmental signals through interlinked genes provides insights into the interactions between fungi and insects. The coordinated optimization of the regulatory networks is necessary for fungi to adapt to their habitats. We demonstrated that the synergistic regulation of sensor histidine kinase (SLN1) and acetyl-CoA carboxylase (ACC1) plays a critical role in regulating the fungal response to Sinella curviseta stress. Furthermore, we found that the enhanced production of trehalose, carotenoids, and 5-MTHF plays crucial role in the resistance to the fungivore. Our results provide insights into the understanding of the adaptation of N. crassa to environmental stimuli.

Exposure to TiO2 nanoparticles improves the physiological characteristics of drought‐challenged chickpeas (Cicer arietinum L.)

AbstractDrought stress markedly affects plant growth and crop production. In turn, treatment with some metal‐based nanoparticles (NPs) such as TiO2‐NPs could improve the plant tolerance against drought stress. In the present study, the effects of different levels of moisture regime (40%, 60%, and 90% field capacity [FC]) in conjunction with various concentrations of TiO2‐NPs (0, 5, 10, 20, and 40 mg. L−1) on chickpea were studied. Exposure of drought‐challenged chickpea plants to TiO2‐NPs raised antioxidant enzyme activity compared with plants grown under drought without TiO2‐NP treatment. The highest activity of ascorbate peroxidase (APX) was observed at 40% FC and application of 40 mg. L−1 TiO2‐NPs. Moreover, peroxidase (POX) activity has increased with the enhancing concentration of TiO2‐NPs to 20 mg. L−1 at 90% FC. In comparison, the application of 40 mg. L−1 TiO2‐NPs and decreasing levels of FC caused a rise in the activity of superoxide dismutase (SOD). Exposure to TiO2‐NPs raised the amount of total phenols and 2,2‐diphenyl‐1‐picryl‐hydrazyl‐hydrate (DPPH) at different levels of moisture regime. The content of malondialdehyde (MDA) at 60% FC has decreased by 22% after treatment with 20 mg. L−1 TiO2‐NPs compared with control plants. Also, treatment with TiO2‐NPs heightened the proline content, and the highest amount of proline was obtained at 40% FC by applying 20 mg. L−1 NPs. The treatment with TiO2‐NPs in the moisture regimes led to higher chlorophyll and carotenoid production in chickpea plants. Taken together, the application of TiO2‐NPs could raise the defense potential of chickpea plants against oxidative stress caused by the generation of reactive oxygen species.

Extraction and Purification of Highly Active Astaxanthin from Corynebacterium glutamicum Fermentation Broth.

The marine carotenoid astaxanthin is one of the strongest natural antioxidants and therefore is used in a broad range of applications such as cosmetics or nutraceuticals. To meet the growing market demand, the natural carotenoid producer Corynebacterium glutamicum has been engineered to produce astaxanthin by heterologous expression of genes from the marine bacterium Fulvimarina pelagi. To exploit this promising source of fermentative and natural astaxanthin, an efficient extraction process using ethanol was established in this study. Appropriate parameters for ethanol extraction were identified by screening ethanol concentration (62.5-97.5% v/v), temperature (30-70 °C) and biomass-to-solvent ratio (3.8-19.0 mgCDW/mLsolvent). The results demonstrated that the optimal extraction conditions were: 90% ethanol, 60 °C, and a biomass-to-solvent ratio of 5.6 mgCDW/mLsolvent. In total, 94% of the cellular astaxanthin was recovered and the oleoresin obtained contained 9.4 mg/g astaxanthin. With respect to other carotenoids, further purification of the oleoresin by column chromatography resulted in pure astaxanthin (100%, HPLC). In addition, a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay showed similar activities compared to esterified astaxanthin from microalgae and a nine-fold higher antioxidative activity than synthetic astaxanthin.

Antioxidant Capacity of Carotenoid Extracts from the Haloarchaeon Halorhabdus utahensis.

Herein, we report on the production, characterization, and antioxidant power assessment of carotenoids from the haloarchaeon Halorhabdus utahensis. It was grown at 37 °C and 180 rpm agitation in halobacteria medium supplemented with glucose, fructose, and xylose, each at concentrations of 0.2%, 1%, and 2%, and the carotenoid yield and composition were investigated. The microorganism produced the carotenoids under all the conditions tested, and their amount followed the order glucose < xylose < fructose. The highest yield was achieved in 2% fructose growth medium with 550.60 ± 7.91 μg/g dry cell and 2428.15 ± 49.33 μg/L. Separation and identification of the carotenoids were performed by RP-HPLC and HPLC/APCI-ITMSn. Bacterioruberin was the main carotenoid detected and accounted for 60.6%, 56.4%, and 58.9% in 2% glucose, 1% xylose, and 2% fructose extracts, respectively. Several geometric isomers of bacterioruberin were distinguished, and representatives of monoanhydrobacterioruberin, and bisanhydrobacterioruberin were also detected. The assignment to cis-isomers was attempted through analysis of the UV/Vis spectra, intensity of cis peaks, and spectral fine structures. The extracts exhibited superoxide scavenging activity higher than butylhydroxytoluene, ascorbic acid, and Trolox, selected as antioxidant references. The anti-hyaluronidase capacity was investigated, and the 2% fructose extract showed the highest activity reaching 90% enzyme inhibition with 1.5 μg. The overall data confirm that Hrd. utahensis can be regarded as an interesting source of antioxidants that can find applications in the food and cosmetic sectors.

Differential gene expression in a subpopulation of Phaeodactylum tricornutum with enhanced growth and carotenoid production after FACS-mediated selection

Fluorescence-Activated Cell Sorting (FACS) is a powerful method with many applications in microalgal research, especially for screening and selection of cells with improved phenotypes. However, the technology requires review of gene expression changes responsible for enhanced phenotypes in sorted populations. Phaeodactylum tricornutum cells were sorted using FACS with excitation/emission parameters targeted to favouring the industrially-relevant carotenoid fucoxanthin. The resulting cultures showed significantly higher growth rate (1.10 ×), biomass (1.30 ×), chlorophyll a levels (1.22 ×) and fucoxanthin content (1.28 ×) relative to the wild-type strain. RNA-seq was used to elucidate the underlying molecular-level regulatory changes associated with these traits and represents the first study do so on FACS-sorted microalgal cultures. Transcriptome analysis corroborated evidence of increased chlorophyll a and fucoxanthin, showing enrichment for the genes/pathways for tetrapyrrole biosynthesis and for suites of genes directly related to photosynthesis. Only three genes were upregulated in the MEP (non-mevalonate) pathway to carotenoid biosynthesis pathway, suggesting either a strong influence of IDI, CRTISO5 and ZEP1 on fucoxanthin biosynthesis or a post-transcriptional or post-translational mechanism for the observed increase in fucoxanthin content.

Production kinetics and characterization of natural food color (torularhodin) with antimicrobial potential

The present investigation was conducted to optimize the fermentative production of food color (carotenoid pigments) using Rhodotorula sp. grown under batch cultivation. The effect of different carbon sources, nitrogen source and pH on carotenoid yield was investigated initially. The maximum carotenoid yield of 3520.23 and 2992.65 μg/g were obtained using sorbitol and beef extract as potential carbon and nitrogen sources, respectively, at pH 7.0. Further, process optimization was done using RSM to enhance the carotenoid yield. The optimized condition comprised; 2.0 g/L sorbitol, 0.32 g/L beef extract and 6.25 pH. Optimized condition scale-up in 7.5 L bioreactor (working volume 3.0 L) gave maximum carotenoid and biomass yield of 7325.70 ± 0.04 μg/g and 0.589 ± 0.05 g/L, respectively, after 48 h of cultivation. Leudking Piret model deduced mixed growth associated carotenoid production. The carotenoid pigment was characterized as torularhodin by FTIR and showed antimicrobial activities against E. coli and S. aureus.

A novel drying film culture method applying a natural phenomenon: Increased carotenoid production by Haematococcus sp

Low productivity and high cost remain major bottlenecks for the large-scale production of Haematococcus sp. This study explored biomass production and carotenoid accumulation in Haematococcus sp. (KCTC 12348BP) using drying film culture. The broth-cultured strain (3.2 × 106 cells/mL, 0.83 ± 0.02 mg/mL for a 21 d culture) was cultured under various conditions (different inoculum volumes and mist feeding intervals) in waterless agar plates at 28 ± 0.5 °C, under fluorescent light (12 h light–dark cycle) for 1 month. The maximum biomass obtained was 17.60 ± 0.72 g/m2, while the maximum astaxanthin concentration was 8.23 ± 1.13 mg/g in the culture using 1 mL inoculum and 3 d feeding interval. Drought stress in drying film culture effectively induced the accumulation of carotenoids from β-carotene, facilitating the production of canthaxanthin via the astaxanthin biosynthesis pathway. This cost-effective culture system can increase the biomass and carotenoid pigment production in Haematococcus sp.