Beta-carotene Production Research Articles

A REVIEW ON THE β-CAROTENE: PRODUCTION, EXTRACTION, SYNTHESIS AND BIOLOGICAL PROPERTIES

The presence of double bonds gives β-carotene its distinctive hue, making it an oxygen-deficient isoprene-containing molecule. There are cyclic rings at both ends of the molecule. The present review was based on the production, extraction, synthesis and biological properties of β-carotene. Mammals can't produce carotenoids from scratch; therefore, they must get them from somewhere else. Algae, higher plants (in fruits and flowers as esters), fungi, and animals all have these yellow, red, and orange pigments in their natural forms. Carotenoids are found in human breast milk and can be modified through the mother's diet. Hexane, tetrahydrofuran, and acetone are examples of aprotic organic solvents that can be used for solvent-based extraction of -carotene from algae after and before they have been treated. The -carotene in algae, fungi, and plants can be extracted with the help of these solvents. The extraction process for -carotene is sped up and improved by the use of supercritical fluids. Increasing beta-carotene production can be accomplished by tailoring a number of environmental factors, including the type of organism used in the cultivation process, the amount of light available, the amount of salt present, the pH level, and the type and availability of carbon sources. Microalgae, plants, and fungus have all been optimised to increase their beta-carotene synthesis. It concluded that when administered in different doses, it has been shown to have effects like antioxidant and anti-inflammatory. it is also effective against cancer, cardiovascular diseases, Covid-19 and various other diseases.

Life cycle assessment of microalgal cultivation medium: biomass, glycerol, and beta-carotene production by Dunaliella salina and Dunaliella tertiolecta

Abstract Purpose Dunaliella is a halophilic genus of microalgae with high potential in the global food market. The microalgal cultivation process contributes to not only economic impact but also environmental impact, especially regarding the artificial medium composition. In this context, a life cycle assessment was carried out to analyze the impacts associated with the components of the modified Johnson medium (MJM) and to predict the best scenarios to cultivate Dunaliella tertiolecta and Dunaliella salina for biomass, glycerol, and beta-carotene production. Method Two chains were analyzed separately: (1) Dunaliella salina (strain DF 15) cultivated in 8 scenarios combining different nitrogen (0.1 and 1.0 g L−1 KNO3) and magnesium (1.1–2.3 g L−1 MgCl2.6H2O) concentrations to produce biomass, glycerol, and beta-carotene and (2) Dunaliella tertiolecta (strain CCAP 19/30) cultivated in 5 scenarios combining different nitrogen (0.1 and 1.0 g L−1 KNO3) and salt (116.9–175.4 g L−1 sea salt) concentrations to produce biomass and glycerol. In addition, we evaluated the potential of cultivating these species to reduce the carbon footprint of the proposed scenarios. Results and discussion For D. salina, S5 (1 g L−1 KNO3, 1.1 g L−1 MgCl2.6H2O) had the lowest environmental damage for biomass (74.2 mPt) and glycerol production (0.95 Pt) and S3 (0.1 g L−1 KNO3, 1.9 g L−1 MgCl2.6H2O) for beta-carotene (3.88 Pt). T4 (1 g L−1 KNO3, 116.9 g L−1 sea salt) was the best for D. tertiolecta for biomass (74 mPt) and glycerol (0.49 Pt). “Respiratory inorganics,” “Non-renewable energy,” and “Global warming” were the most impacted categories. “Human health,” “Climate change,” and “Resources” had the highest share of all damage categories. All the scenarios presented negative carbon emission after proposing using brine as alternative salt source: S5 was the best scenario (− 157.5 kg CO2-eq) for D. salina and T4 for D. tertiolecta (− 213.6 kg CO2-eq). Conclusion The LCA proved its importance in accurately predicting the optimal scenarios for MJM composition in the analyzed bioproducts, as confirmed by the Monte Carlo simulation. Although the absolute values of impacts and productivity cannot be directly compared to large-scale cultivation, the validity of the LCA results at this scale remains intact. Productivity gains could outweigh the impacts of “surplus” MJM components. Our study showcased the potential of combining D. salina and D. tertiolecta cultivation with CO2 capture, leading to a more environmentally friendly cultivation system with a reduced carbon footprint.

Evaluation of effective environmental parameters on lipid, protein and beta-carotene production in Spirulina platensis microalga

Microalgae are able to produce various products, including biofuel, proteins, and beta-carotene. Hence, the determination of optimal conditions for the production of these compounds is of utmost importance. This study aims to evaluate the impact of environmental parameters on the production of lipid, protein, and beta-carotene in Spirulina platensis microalgae. The studied parameters included salinity levels of 1, 2, and 3 M, pH values above and below seven, nitrate concentrations of 0.25 and 0.5 g L−1, light intensities of 2500 and 5000 lux, and temperatures of 25 and 30 °C. The results suggest that the highest protein level is synthesized at 2 M salinity, acidity above seven, 0.25 g L−1 nitrate concentration, 5000 lux of light intensity, and 30 °C temperature. However, the optimal conditions for lipid accumulation are found as the salinity of 2 M, pH greater than 7, nitrate concentration of 0.25 g L−1, the light intensity of 5000 lux, and temperature of 30 °C. The optimal beta-carotene production occurs at 3 M salinity, pH ≥ 7, nitrate concentration of 0.5 g L−1, the light intensity of 5000 lux, and temperature of 30 °C. A better understanding of the optimal conditions can ensure the maximum production of bioproducts, including biofuel, protein, and beta-carotene in Spirulina platensis microalga, at minimum costs.

Optogenetic control of beta-carotene bioproduction in yeast across multiple lab-scales.

Optogenetics arises as a valuable tool to precisely control genetic circuits in microbial cell factories. Light control holds the promise of optimizing bioproduction methods and maximizing yields, but its implementation at different steps of the strain development process and at different culture scales remains challenging. In this study, we aim to control beta-carotene bioproduction using optogenetics in Saccharomyces cerevisiae and investigate how its performance translates across culture scales. We built four lab-scale illumination devices, each handling different culture volumes, and each having specific illumination characteristics and cultivating conditions. We evaluated optogenetic activation and beta-carotene production across devices and optimized them both independently. Then, we combined optogenetic induction and beta-carotene production to make a light-inducible beta-carotene producer strain. This was achieved by placing the transcription of the bifunctional lycopene cyclase/phytoene synthase CrtYB under the control of the pC120 optogenetic promoter regulated by the EL222-VP16 light-activated transcription factor, while other carotenogenic enzymes (CrtI, CrtE, tHMG) were expressed constitutively. We show that illumination, culture volume and shaking impact differently optogenetic activation and beta-carotene production across devices. This enabled us to determine the best culture conditions to maximize light-induced beta-carotene production in each of the devices. Our study exemplifies the stakes of scaling up optogenetics in devices of different lab scales and sheds light on the interplays and potential conflicts between optogenetic control and metabolic pathway efficiency. As a general principle, we propose that it is important to first optimize both components of the system independently, before combining them into optogenetic producing strains to avoid extensive troubleshooting. We anticipate that our results can help designing both strains and devices that could eventually lead to larger scale systems in an effort to bring optogenetics to the industrial scale.

Dunaliella salina Biorefinary: An Evaluation of the State-of-the-Art

Biorefinery is an important concept that can be applied to improve the industrial processes, making them more sustainable, and reducing waste, and costs of operation. Algal biomass is too onerous to be exploited, and only a few microalgae have products that are feasible to commercialize. Combining this concept and other ones, such as intensification and integration procedures, can also help reduce the costs and become more attractive to industries. Dunaliella salina is one of the most critical microalgae studied nowadays. Currently, its process exploits only 10% of its compounds in beta-carotene production. Studies show that different compounds can be exploited and the processes can be combined in a biorefinery approach.

Novel carotenogenic gene combinations from red yeasts enhanced lycopene and beta-carotene production in Saccharomyces cerevisiae from the low-cost substrate sucrose.

Carotenoids (C40H56) including lycopene and beta-carotene are relatively strong antioxidants that provide benefits to human health. Here, we screened highly efficient crt variants from red yeasts to improve lycopene and beta-carotene production in Saccharomyces cerevisiae. We identified that crt variants from Sporidiobolus pararoseus TBRC-BCC 63403 isolated from rice leaf in Thailand exhibited the highest activity in term of lycopene and beta-carotene production in the context of yeast. Specifically, the phytoene desaturase SpCrtI possessed up to 4-fold higher in vivo activity based on lycopene content than the benchmark enzyme BtCrtI from Blakeslea trispora in our engineered WWY005 strain. Also, the geranylgeranyl pyrophosphate (GGPP) synthase SpCrtE, the bifunctional phytoene synthase-lycopene cyclase SpCrtYB, and SpCrtI when combined led to 7-fold improvement in beta-carotene content over the benchmark enzymes from Xanthophyllomyces dendrorhous in the laboratory strain CEN.PK2-1C. Sucrose as an alternative to glucose was found to enhance lycopene production in cells lacking GAL80. Lastly, we demonstrated a step-wise improvement in lycopene production from shake-flasks to a 5-L fermenter using the strain with GAL80 intact. Altogether, our study represents novel findings on more effective crt genes from Sp. pararoseus over the previously reported benchmark genes and their potential applications in scale-up lycopene production.

Potential of a mixed culture of microalgae for accumulation of beta-carotene under different stress conditions

Abstract Beta-carotene, a pigment found in plants, is mainly produced by microalgae. Nevertheless, this production has only been investigated in pure cultures. Beta-carotene production through mixed culture eliminates the costly procedure of sterilization and contamination prevention needed for pure cultures. In this study, for the first time, the growth, beta-carotene, and chlorophyll production of a mixed culture of microalgae from Caspian Sea was investiagted under different stress conditions. At the condition of tripled light intensity and nitrogen starvation, beta-carotene content increased from 18.03 to 43.8 and 46.5 mol beta-carotene g−1 protein, respectively. However, the salinity of 4 mol L−1 caused the beta-carotene content to fall to zero. The blank sample reached a constant value of 23 mol beta-carotene g−1protein. The comparable results with the specific monoculture species exhibit the high potential of a mixed culture of microalgae for beta-carotene production without need of the high sterilization cost. Nevertheless, more research is needed for where it can be a good substitute for pure culture.

The effect of audible sound frequency on the growth and beta-carotene production of Dunaliella salina

Sound vibrations as a cutting-edge technology considered for promoting the growth of microalgae. In this experiment, the authors have carried out a pilot study to observe the response of Dunaliella salina to audible sound frequencies (100, 200, 500, 1000 Hz). The growth and beta-carotene concentration as a secondary metabolite in optimal culture medium and sub-optimal medium with nitrate deficiency were investigated. The control was in an acoustic enclosure without any sound frequency exposure and cultured in the N-sufficient medium. The sound frequency of 200 Hz was found to promote biomass concentration and cell division up to 50% compared to control. Interestingly, the frequency of 200 Hz promoted growth when the nitrate concentration decreased by 75% in the culture medium. The simultaneous effect of audible sound frequency of 200 Hz and nitrate deficiency showed the amount of beta-carotene increased to 37% compared to control. In the absence of sound frequency, an increase of 18% in beta-carotene was observed. To the best of our knowledge, the frequency of 200 Hz is a good demonstration of the ability of sound stimuli to improve the growth as well as a secondary metabolite of D.salina.

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The effect of different light spectra on beta-carotene production by Dunaliella salina

This study focused on the effect of different light intensities and spectra on the beta-carotene production within Dunaliella salina cells (green eukaryote microalgae) which are purified from Urmia Lake in the northwest of Iran. For this purpose, four LED light spectra (white light: 360–760 nm, red light: 620–645 nm, yellow light: 587–595 nm, blue light: 460–475 nm) were used in this experimental research. The light intensity of 200 µmol/(m2 s) was considered for each the LED light spectra. The highest beta-carotene content extracted under a sequential combination of colored light (white, blue, red and yellow respectively) was 16.93 µg of beta-carotene per mg of cell dry weight and the highest accumulated beta-carotene within the cells among single-colored light was 15.16 µg /mg when the cells were cultivated under yellow light.

The Effect of Instantaneous and Slow Release Salt Stress Methods on Beta-carotene Production within Dunaliella Salina Cells

The main concept of the present study is taken from the growth pattern of Dunaliella salina (green unicellular eukaryote microalgae) in Urmia Lake which is the second biggest saline lake in the world. In this study, different models of salt stress, including instantaneous (1 and 2 M) and consecutive slow-release salt stresses (0.5 M) were tested, and the amount of beta-carotene for each test was evaluated. According to the results, the highest amount of beta-carotene was obtained by the slow release method of salt injection with the amount of 9.01 µg of beta-carotene per mg of the dry weight of microalgae. The largest amount of beta-carotene production in 1 and 2 M instantaneous salt stresses method were recorded as 4.35 and 0.65 µg mg-1 respectively which were up to 2 and 14 times lower than the highest beta-carotene production under slow-release salt stress method.

Factors affecting production of beta-carotene from Dunaliella salina microalgae

Abstract Dunaliella Salina is a carotene-producing microalgae well-known for its superb ability to accumulate valuable beta-carotene. The factors affecting the bio-beta-carotene production from Dunaliella salina microalgae have been investigated in this study. First, the different usages of Dunaliella salina, especially for the production of beta-carotene, are described. Then, the factors affecting the process of obtaining beta-carotene from Dunaliella salina in the culture system, culture conditions, and downstream processes are investigated. Investigations show that application of airborne photo-bioreactor culture system improves the mechanism of production. It was found that among the culture conditions, the parameters such as ambient salinity, temperature, and intensity of illumination are the most effective ones. The optimal values for these parameters, the highest cellular density of Dunaliella salina and the highest beta-carotene production are determined. The extraction step is known as the most critical step in the downstream processes of beta-carotene production.

Towards a sustainable Dunaliella salina microalgal biorefinery for 9-cis β-carotene production

Valorisation of the efficacy of 9-cis beta-carotene in treating atherosclerosis, psoriasis, and inhibiting atherogenesis and retinitis pigmentosa is becoming increasingly urgent, but supplies of 9-cis beta-carotene are scarce and this compound is difficult to synthesise chemically, unlike the much more common all-trans form. Innovative products, processes and services in an algal biorefinery that rely on renewable biological resources instead of fossil fuel alternatives offer the potential to lower the energy costs of traditional chemical processes and reduce carbon emissions, water usage and waste. In 2013, the European Commission supported development of 4 microalgal biorefinery projects to assess the potential for innovative approaches to tackle the major challenges intrinsic to the development of the algae biorefineries. One of these was the D-Factory (KBBE.2013.3.2-02) which sought to evaluate requirements for sustainable, industrial-scale production of Dunaliella salina and extraction of its carotenoids, especially 9-cis beta-carotene in a CO2 microalgae biorefinery. Here we present findings of the D-Factory project and propose a way forward for industrial-scale production of 9-cis beta-carotene using biotechnology based on Dunaliella salina biomass. Cultivation improvements are able to deliver more than double the current levels of productivity, with increased sustainability, whilst the use of natural hyper-accumulating carotenogenic strains combined with the use of red light to boost production of the beta-carotene pathway, will increase the relative concentration of 9-cis beta-carotene in extracts of carotenoids with consequent improvements in downstream processing. These developments pave the way for acquiring data for a Medicine Licence and prepare the market for entry of novel 9-cis beta-carotene products.

Engineering yeast artificial core promoter with designated base motifs

BackgroundSynthetic biology requires toolbox of promoters to finely tune gene expression levels for building up efficient cell factories. Yeast promoters owned variable core promoter regions between the TATA-box and transcriptional starting site (TSS) at the length mostly around 20–80 bases. This region allowed flexible design of artificial promoter but potentially demand special base motifs to maintain or enhance the promoter’s strength.ResultsHere, we designed and screened the base motifs and tested the activities of yeast artificial core promoters. Different 30 bases of artificial sequences led to variable expression levels of CrtY enzyme which determined the lycopene–carotene compositions, represented in the colony-color spectrum of red–orange–yellow. The upstream sequences of two strong promoter PEXP1 and PGPD and two starting strains with distinguishable lycopene production levels were utilized to characterize the promoter sequences. Different partition designs of T-rich or G/C-rich base motifs led to distinguishable colony-color distributions. Finally, we screened a champion promoter with a highest 5.5-fold enhancement of lycopene–carotene transformation. Another selected promoter generated a highest beta-carotene production as 7.4 mg/g DCW.ConclusionsThis work offered an approach to redesign promoter with artificial sequences. We concluded that the core promoter region could be designated as 30 bases and different base motifs would enhance or weaken the promoter’s strength. Generally, more T-rich elements, higher %T and lower G/C percentage were beneficial to enhance the strength of artificial core promoter.

Dual production of polyunsaturated fatty acids and beta-carotene with Mucor wosnessenskii by the process of solid-state fermentation using agro-industrial waste

Solid-state fermentation is a technique employing microorganisms grown on a solid substrate in the absence of free water. The substrates used in this process are mostly waste from the agro-industry (brans, spent malt grains, distiller grains, etc.) that improves not only the economy of the process but also has positive effect on waste management problems. Zygomycetous fungi are not only able to grow in such conditions but also enrich fermented materials with various types of bioactive compounds. Mucor sp. strains have been identified as producers of gamma-linolenic acid and beta-carotene in submerged fermentation. The aim of the present study was to identify the best microbial producer of gamma-linolenic acid and beta-carotene among four different Mucor strains and to study the requirements for the dual production of these metabolites. Mucor wosnessenskii was identified as the most suitable producer of both metabolites. After optimization of the fermentation conditions, the highest yields obtained were 10.7 g of gamma-linolenic acid/kg of fermented product and 261.5 mg of beta-carotene/kg of fermented product. This yield of beta-carotene is the highest among the results published so far.

Assessment, monitoring and modelling of the abundance of Dunaliella salina Teod in the Meighan wetland, Iran using decision tree model.

The microalga Dunaliella salina has been broadly studied for different purposes such as beta-carotene production, toxicity assessment and salinity tolerance, yet research on the habitat suitability of this alga has rarely been reported. The present research aims to apply a suitable monitoring and modelling methods (two critical steps in ecological researches) to predict the abundance of D. salina. The abundance of D. salina was predicted by decision tree model (J48 algorithm) in 10 different monitoring sites during 1-year study period (2016-2017) in the Meighan wetland, one of the valuable hypersaline wetlands in Iran. The abundance of alga (as output of model) together with various water quality and physical-habitat wetland characteristics (as inputs of model) were monthly and repeatedly monitored in two different depths (one from the surface layer and another one from the depth of maximum 50cm) which in total resulted in 240 instances (120 instances for each depth). Based on trial and error, a sevenfold cross-validation resulted in the highest predictive performances of the model (CCI > 75% and Cohen's Kappa > 0.65). According to the model's prediction, the number of sunny hours might be one of the most important driving parameters to meet the habitat requirements of alga in the hypersaline wetland. Model also predicted that an increase in dissolved oxygen and sodium concentrations might increase the abundance of D. salina in the salt wetland. In contrast, an increase in total suspended solids concentration and monthly precipitation might lead to a decrease in the abundance of alga. Chi-square test of independence showed a significant difference between the abundance of the D. salina and spatio-temporal patterns in the wetland (Pearson chi-square statistic = 221.7, p= 0.001) so warm seasons (spring and summer) had more contribution to the sampling of the species than cold seasons (autumn and winter). The difference in the abundance of the species in different sampling sites can be attributed due to the various anthropogenic activities.

Exploiting the Feedstock Flexibility of the Emergent Synthetic Biology Chassis Vibrio natriegens for Engineered Natural Product Production.

A recent goal of synthetic biology has been to identify new chassis that provide benefits lacking in model organisms. Vibrio natriegens is a marine Gram-negative bacterium which is an emergent synthetic biology chassis with inherent benefits: An extremely fast growth rate, genetic tractability, and the ability to grow on a variety of carbon sources (“feedstock flexibility”). Given these inherent benefits, we sought to determine its potential to heterologously produce natural products, and chose beta-carotene and violacein as test cases. For beta-carotene production, we expressed the beta-carotene biosynthetic pathway from the sister marine bacterium Vibrio campbellii, as well as the mevalonate biosynthetic pathway from the Gram-positive bacterium Lactobacillus acidophilus to improve precursor abundance. Violacein was produced by expressing a biosynthetic gene cluster derived from Chromobacterium violaceum. Not only was V. natriegens able to heterologously produce these compounds in rich media, illustrating its promise as a new chassis for small molecule drug production, but it also did so in minimal media using a variety of feedstocks. The ability for V. natriegens to produce natural products with multiple industrially-relevant feedstocks argues for continued investigations into the production of more complex natural products in this chassis.

Media optimization using Box Behnken design for enhanced production of biomass, beta-carotene and lipid from Dunaliella salina

Dunaliella salina a halotolerant microalga is well known for a high fatty acid and beta-carotene content, which makes it a potent source at a commercial level. The current study focuses on optimizing commercially known media using Box Behnken design to attain higher yields of biomass, beta-carotene, and lipids simultaneously. The optimal medium conditions as per response surface methodology were glucose, potassium nitrate, sodium chloride at a concentration of 13.23 g/L, 3.145 g/L and 35.6 g/L, respectively while maintaining the concentration of other nutrients unchanged. Maximum yield of biomass, beta-carotene and lipid productivity attained experimentally using the optimized media was 1.24 g/L, 6.07 mg/g and 20.7 mg/L/day, respectively than their original values i.e., 0.571 g/L of biomass, 4.18 mg/g of beta-carotene and 13.2 mg/L/day of lipid content. Biomass yield was increased by 2.17 folds, beta-carotene and lipid were increased by 1.45 folds and 1.56 folds, respectively.

Algal cell response to laboratory-induced cadmium stress: a multimethod approach.

We examined the response of algal cells to laboratory-induced cadmium stress in terms of physiological activity, autonomous features (motility and fluorescence), adhesion dynamics, nanomechanical properties, and protein expression by employing a multimethod approach. We develop a methodology based on the generalized mathematical model to predict free cadmium concentrations in culture. We used algal cells of Dunaliella tertiolecta, which are widespread in marine and freshwater systems, as a model organism. Cell adaptation to cadmium stress is manifested through cell shape deterioration, slower motility, and an increase of physiological activity. No significant change in growth dynamics showed how cells adapt to stress by increasing active surface area against toxic cadmium in the culture. It was accompanied by an increase in green fluorescence (most likely associated with cadmium vesicular transport and/or beta-carotene production), while no change was observed in the red endogenous fluorescence (associated with chlorophyll). To maintain the same rate of chlorophyll emission, the cell adaptation response was manifested through increased expression of the identified chlorophyll-binding protein(s) that are important for photosynthesis. Since production of these proteins represents cell defence mechanisms, they may also signal the presence of toxic metal in seawater. Protein expression affects the cell surface properties and, therefore, the dynamics of the adhesion process. Cells behave stiffer under stress with cadmium, and thus, the initial attachment and deformation are slower. Physicochemical and structural characterizations of algal cell surfaces are of key importance to interpret, rationalize, and predict the behaviour and fate of the cell under stress in vivo.

Blue-Red LED wavelength shifting strategy for enhancing beta-carotene production from halotolerant microalga, Dunaliella salina.

In the present study, to improve the photosynthetic betacarotene productivity of Dunaliella salina, a blue-red LED wavelength-shifting system (B-R system) was investigated. Dunaliella salina under the B-R system showed enhanced density and beta-carotene productivity compared to D. salina cultivated under single light-emitting diode light wavelengths (blue, white, and red light-emitting diode). Additionally, we developed blue light-adapted D. salina (ALE-D. salina) using an adaptive laboratory evolution (ALE) approach. In combination with the B-R system applied to ALE-D. salina (ALE B-R system), the beta-carotene concentration (33.94 ± 0.52 μM) was enhanced by 19.7% compared to that observed for the non-ALE-treated wild-type of D. salina (intact D. salina) under the B-R system (28.34 ± 0.24 μM).