Enhancement of carotenoids and lipids production by oleaginous red yeast Sporidiobolus pararoseus KM281507

Bioconversion of biodiesel-derived crude glycerol into carotenoids and lipids was investigated by a microbial conversion of an oleaginous red yeast Sporidiobolus pararoseus KM281507. The methanol content in crude glycerol (0.5%, w/v) did not show a significant effect on biomass production by strain KM281507. However, demethanolized crude glycerol significantly supported the production of biomass (8.64?±?0.13?g/L), lipids (2.92?±?0.03?g/L), β-carotene (15.76?±?0.85?mg/L), and total carotenoids (33.67?±?1.28?mg/L). The optimal conditions suggested by central composite design were crude glycerol concentration (55.04?g/L), initial pH of medium (pH 5.63) and cultivation temperature (24.01°C). Under these conditions, the production of biomass, lipids, β-carotene, and total carotenoids were elevated up to 8.83?±?0.05, 4.00?±?0.06?g/L, 27.41?±?0.20, and 53.70?±?0.48?mg/L, respectively. Moreover, an addition of olive oil (0.5???2.0%) dramatically increased the production of biomass (14.47?±?0.15?g/L), lipids (6.40?±?0.09?g/L), β-carotene (54.43?±?0.95?mg/L), and total carotenoids (70.92?±?0.51?mg/L). The oleic acid content in lipids was also increased to 75.1% (w/w) of total fatty acids, indicating a good potential to be an alternative biodiesel feedstock. Meanwhile, the β-carotene content in total carotenoids was increased to 76.7% (w/w). Hence, strain KM281507 could be a good potential source of renewable biodiesel feedstock and natural carotenoids.     

Carotenoids production in different culture conditions by Sporidiobolus pararoseus

Carotenoids produced by Sporidiobolus pararoseus were studied. It was found that biomass was connected with carbon source, temperature, and pH, but carotenoids proportion was seriously influenced by dissolved oxygen and nitrogen source. Different carotenoids could be obtained by using selected optimum conditions. In the end we established the strategies to produce β-carotene or torulene. Fed-batch fermentation in fermentor was used to prove the authenticity of our conclusions. The cell biomass, β-carotene content, and β-carotene proportion could reach 56.32?g/L, 18.92?mg/L and 60.43%, respectively, by using corn steep liquor at 0-5% of dissolved oxygen saturation. β-Carotene content was 271% higher than before this addition. The cell biomass, torulene content, and torulene proportion could reach 62.47?g/L, 31.74?mg/L, and 70.41%, respectively, by using yeast extract at 30-35% of dissolved oxygen saturation. Torulene content was 152% higher than before this addition. The strategy for enhancing specific carotenoid production by selected fermentation conditions may provide an alternative approach to enhance carotenoid production with other strains.     

CAROTENOIDS PRODUCTION IN DIFFERENT CULTURE CONDITIONS BY Sporidiobolus pararoseus

Carotenoids produced by Sporidiobolus pararoseus were studied. It was found that biomass was connected with carbon source, temperature, and pH, but carotenoids proportion was seriously influenced by dissolved oxygen and nitrogen source. Different carotenoids could be obtained by using selected optimum conditions. In the end we established the strategies to produce β-carotene or torulene. Fed-batch fermentation in fermentor was used to prove the authenticity of our conclusions. The cell biomass, β-carotene content, and β-carotene proportion could reach 56.32 g/L, 18.92 mg/L and 60.43%, respectively, by using corn steep liquor at 0–5% of dissolved oxygen saturation. β-Carotene content was 271% higher than before this addition. The cell biomass, torulene content, and torulene proportion could reach 62.47 g/L, 31.74 mg/L, and 70.41%, respectively, by using yeast extract at 30–35% of dissolved oxygen saturation. Torulene content was 152% higher than before this addition. The strategy for enhancing specific carotenoid production by selected fermentation conditions may provide an alternative approach to enhance carotenoid production with other strains.     

Spectroscopy analysis for simultaneous determination of lycopene and β-carotene in fungal biomass of Blakeslea trispora

Blakeslea trispora is a good alternative source for producing such carotenoids as lycopene and β-carotene. The objective of this research was to elaborate a method for the simultaneous determination of lycopene and β-carotene in Blakeslea trispora products using a usual UV-vis spectrophotometer. The standard solutions of the mixture of different concentrations of β-carotene and lycopene were measured with the UV-vis method and correlation formula for the extinction coefficients of 1% standard solution of lycopene in the solvent (hexane) and the ratios of the optical densities at the character peaks of 470 and 502 nm was elaborated. This gives a possibility to calculate the concentrations of lycopene and β-carotene in the mixture. The prediction quality of the UV-vis method was sufficient and the obtained results were very close to the ones, being measured with the HPLC technique. The proposed method can be used for both routine industrial work and academic research, providing the rapid analysis for simultaneous measurements of lycopene and β-carotene.     

Bio-utilization of fruits and vegetables waste to produce β-carotene in solid-state fermentation: Characterization and antioxidant activity

Fruits and vegetable processing industries produce huge waste in the form of peels, seeds, liquid, and molasses which are a good source of carbohydrates, proteins, fibres, vitamins, and minerals. This waste can be utilized for the production of biocolors using fermentation. Utilization of this waste not only valorizes waste disposal problems but also eliminate environmental pollution. The aim of the present work was to extract and optimize environmental factors for production of β-carotene from fruits and vegetable waste (orange, carrot, and papaya peels) using microbial strain Blakeslea trispora (+) MTCC 884 in solid state fermentation. It was observed that the production of β-carotene was significantly influenced by varying all factors and gave a maximum yield of 0.127 mg/mL. Characterization of the extracted color was done with techniques like HPLC, LCMS, FTIR and Mass Spectroscopy. Mass spectroscopy of extracted color represented the m/z value 537.608 and LCMS analysis gave the eluted peaks at (Rt 13.37), which confirmed the presence of β-carotene. Furthermore, β-carotene percentage estimated by HPLC and LCMS was over 76% suggesting that these fruits and vegetable wastes may be used for the production of β-carotene with high purity and gave good antioxidant properties as determined by DPPH and ABTS.     

Characterization of an evolved carotenoids hyper-producer of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> through bioreactor parameter optimization and Raman spectroscopy

An evolutionary engineering approach for enhancing heterologous carotenoids production in an engineered Saccharomyces cerevisiae strain was used previously to isolate several carotenoids hyper-producers from the evolved populations. β-Carotene production was characterized in the parental and one of the evolved carotenoids hyper-producers (SM14) using bench-top bioreactors to assess the impact of pH, aeration, and media composition on β-carotene production levels. The results show that with maintaining a low pH and increasing the carbon-to-nitrogen ratio (C:N) from 8.8 to 50 in standard YNB medium, a higher β-carotene production level at 25.52 ± 2.15 mg β-carotene g^?1 (dry cell weight) in the carotenoids hyper-producer was obtained. The increase in C:N ratio also significantly increased carotenoids production in the parental strain by 298 % [from 5.68 ± 1.24 to 22.58 ± 0.11 mg β-carotene g^?1 (dcw)]. In this study, it was shown that Raman spectroscopy is capable of monitoring β-carotene production in these cultures. Raman spectroscopy is adaptable to large-scale fermentations and can give results in near real-time. Furthermore, we found that Raman spectroscopy was also able to measure the relative lipid compositions and protein content of the parental and SM14 strains at two different C:N ratios in the bioreactor. The Raman analysis showed a higher total fatty acid content in the SM14 compared with the parental strain and that an increased C:N ratio resulted in significant increase in total fatty acid content of both strains. The data suggest a positive correlation between the yield of β-carotene per biomass and total fatty acid content of the cell.     

溶氧对Blakeslea trispora分批发酵生产β-胡萝卜素的影响

在摇瓶和5 L发酵罐中研究了溶氧 (DO) 对Blakeslea trispora分批发酵生产β-胡萝卜素的影响,总结了5 L发酵罐中β-胡萝卜素发酵过程中溶氧的变化规律.结果表明,当500 mL摇瓶装液量为50 mL,转速为240 r/min条件下发酵生产β-胡萝卜素产量最大,达到3.416 g/L; 5 L发酵罐中,在搅拌转速为1 000 r/min,通气量为1.5 vvm的条件下,β-胡萝卜素的产量可达到3.712 g/L,略高于摇瓶,这可能是由于5 L发酵罐中的气液传递和混合状况好于摇瓶,促进了产物的合成.     

Effect of Biomass Pre-Treatment and Solvent Extraction on β-Carotene and Lycopene Recovery from Blakeslea trispora Cells

Abstract

The production of carotenoids from Blakeslea trispora cells in a synthetic medium has been reported, with the main products being β-carotene, lycopene, and γ-carotene. The effect of biomass pretreatment and solvent extraction on their selective recovery is reported here. Eight solvents of class II and III of the International Conference of Harmonization: ethanol, methanol, acetone, 2-propanol, pentane, hexane, ethyl acetate, and ethyl ether, and HPLC analysis were used for the evaluation of their selectivities towards the three main carotenoids with regard to different biomass pre-treatment. The average C_max values (maximum concentration of caronoids in a specific solvent) were estimated to 16 mg/L with the five out of eight solvents investigated, whereas methanol, pentane, and hexane gave lower values of 10, 11, and 9 mg/L, respectively. The highest carotenoid yield was obtained in the case of wet biomass, where 44–56% is recovered with one solvent and three extractions and the rest is recovered only after subsequent treatment with acetone; thus, four extractions of 2.5 h are needed. Two extractions of 54 min are enough to recover carotenoids from dehydrated biomass, with the disadvantage of a high degree of degradation. Our results showed that, for maximum carotenoid recovery, ethyl ether, 2-propanol, and ethanol could be successfully used with biomass without prior treatment, whereas fractions enriched in β-carotene or lycopene can be obtained by extraction with the proper solvent, thus avoiding degradation due to time-consuming processes.     

Profiling of carotenoids in six microalgae (Eustigmatophyceae) and assessment of their β-carotene productions in bubble column photobioreactor

The profiles of carotenoids and production of β-carotene by six eustigmatophytes, Eustigmatos magnus, Eustigmatos polyphem, Eustigmatos vischeri, Vischeria helvetica, Vischeria punctata and Vischeria stellata, grown in a bubble column photobioreactor were measured. All eustigmatophytes contained β-carotene, violaxanthin and vaucheriaxanthin as their major carotenoids and accumulated large amount of β-carotene, which accounted for over 50?% of total carotenoids. Maximum intracellular β-carotene contents ranged 1.5–3.5?% of dry wt and in V. stellata it reached 5.9?% dry wt, accompanied by a biomass dry wt >7.3?g/l, with the highest up to 9.8?g/l. These eustigmatophytes are thus promising producers of β-carotene.     

Effect of the ratio of (+) and (−) mating type of Blakeslea trispora on carotene production from cheese whey in submerged fermentation

The effect of the ratio of (+) and (?) mating type of Blakeslea trispora on carotene production from deproteinized hydrolysed whey in shake flask culture was investigated. Also, the inoculum ratio of the two mating types on the morphology of the microorganism and the relationship between morphological changes of the fungus and product formation were studied. The concentration of carotenes was significantly affected by the ratio of (+) and (?) mating type of B. trispora. A ratio of 1:10 up to 1:100 of (+) and (?) type was found to achieve the highest carotene yields. The optimum ratio of the (+) and (?) mating types for the maximum pigment production (175.0 mg/g dry biomass at 8 days of fermentation) was found to be 1:10. The carotene content in B. trispora consisted of β-carotene, γ-carotene, and lycopene. At the maximum concentration of carotenes the proportion of β-carotene, γ-carotene, and lycopene (as percent of total carotenes) was 80, 12, and 8%, respectively. B.trispora growing in submerged fermentation is able to develop complex morphologies which have been classified into three major groups: freely dispersed hyphae, clumps and pellets. These parameters are strongly influence the production of carotenes.     

氮源种类及溶氧水平对锁掷酵母产类胡萝卜素组分的影响

在锁掷酵母（Sporidioboluspararoseus）发酵产类胡萝卜紊的过程中,发酵产物中类胡萝卜紊种类繁多,而且性质相似,加大了不同色素分离纯化的难度。为定向积累不同种类的类胡萝卜素,以本实验室保藏锁挪酵母JD-2为出发菌,研究了氮源种类和浓度及溶氧对锁掷酵母产类胡萝卜素的影响,并在7L发酵罐中进行了补料分批发酵试验。发现培养基中同时添加有机氮源和无机氮源且溶氧控制较低（5％）时有利于β-胡萝卜素的大量积累,最佳有机氮源和无机氮源分别为玉米浆（20g／L）、硫酸铵（5g／L）。补料分批发酵时β-胡萝卜素产量达到31．28mg／L,红酵母烯12．38mg／L。培养基中只添加有机氮源且相对溶氧控制相对较高（30％）时有利于红酵母烯的大量积累,最佳有机氮源为酵母膏（20g／L）。补料分批发酵时红酵母烯产量达到38．96mg／L,8．胡萝卜素12．36mg／L。     

Activation of the biosynthesis of carotenoids by Blakeslea trispora

The research carried out by several scientists has made possible the industrial preparation of β-carotene by fermentation. A fungus, Blakeslea trispora, abundantly synthesizes carotenoids when its two opposite forms are cultivated together in a special fatty medium. When ionones or other natural substances are introduced into the culture, a very obvious increase in the biosynthesis of carotenoids, more specifically of β-carotene, is obtained. Our own work has shown that; (1) several synthetic products chemically related to β-ionone, such as 2,6,6-trimethyl-l-acetyleyelohexene, can advantageously replace either partially or totally the ionones as inductors of the biosysnthesis of β-carotene; (2) various nitrogen-containing substances when added to the culture medium can considerably enhance the biosysnthesis of carotenoids while sometimes very specically orienting it. Their action comes on top of that of the ionones or their substitutes; actually this action is unexplained. Thus certain amides, imides, lactams, hydrazides, or substituted pyradines, and in particular succinimide and isonicotinoylhydrazine, have produced a two or threefold increase in the quantity of β-carotene present in the culture media of Blackeslea trispora. Conversely some heterocyclic substances such as pyridine itself or imidazole totally inhibit the biosysnthesis of β-carotene but induce the production of very important quantities of lycopene.     

Production kinetics of β-carotene from Planococcus sp. TRC1 with concomitant bioconversion of industrial solid waste into crystalline cellulose rich biomass

Cost effective bioprocessing of nutraceuticals in present global scenario is a matter of concern. This study explored Paper mill sludge (PMS) and sugarcane bagasse (SCB) as inexpensive substrate for Planococcus sp. TRC1 mediated valuable β-carotene production and residual treated biomass as value added crystalline cellulose source simultaneously. Both biomass supported significant bacterial growth reaching highest yield 38.54 ± 1.4 mg/g on PMS (36 h) and 47.13 ± 1.9 mg/g (48 h) on SCB in solid state fermentation. Luedeking-Piret model revealed growth associated production with α and much lower β values of 5.18 and 0.24 for PMS and 4.5 and 0.165 for SCB. Cost analysis exhibited decrementation of pigment cost/mg by 84 % compared to synthetic media. Optimum production conditions were 30 °C temperature, pH 7, 10 % inoculum and initial moisture content 80 % (PMS) and 85 % (SCB). TLC (R_f = 0.9), HPLC (RT = 7.646) and lambda max (465 nm) confirmed pigment’s β-carotene nature with significant antioxidant and antimicrobial activity. It showed stability at varied temperature, pH and light conditions along with negligible phytotoxicity on Vigna radiata. Planococcus sp. TRC1 delignified PMS (41 %) and SCB (38 %) and FT-IR, FESEM and XRD suggested crystalline nature of residual cellulose rich fraction shedding light on a biorefinery approach for valorization of industrial solid wastes.     

Production of carotenoids by strains of Rhodotorula glutinis cultured in raw materials of agro-industrial origin

The production of carotenoids by strains of Rhodotorula glutinis on different raw materials of agro-industrial origin (grape must, glucose syrup, beet molasses, soybean flour extract, maize flour extract) was investigated. Maximum yield (5.95 mg/l of total carotenoids culture fluid, 630 μg/g dry cell weight) was obtained with a particular strain of Rhodotorula glutinis after a batch culture of 120 h in a substrate containing concentrated rectified grape must as the sole carbohydrate source. In all experiments, the major pigments forming carotenoids (β-carotene, torulene, torularhodin) were quantified.     

Optimization of carotenoid production by Rhodotorula graminis DBVPG 7021 as a function of trace element concentration by means of response surface analysis

As the final step of a study aiming at the optimization of culture conditions for the production of carotenoids by red yeasts, a statistically-based experimental design has been applied to assess the influence of selected trace elements on carotenogenesis in Rhodotorula graminis DBVPG 7021. In particular, a central composite design scheme has been used to evaluate the influence of Fe³⁺, Co²⁺, Mn²⁺, Al²⁺ and Zn²⁺ (within the range 0–50 ppm) on various responses, namely biomass (B), total carotenoid production (TC) and percentage of specific carotenoids (β-CAR, β-carotene; γ-CAR, γ-carotene; TN, torulene; TD, torularhodin) on total carotenoids. Second-order polynomial models were calculated and reduced equations were designed by neglecting non-significant (P < 0.01) regression coefficients. Reduced equations were used to calculate the optimal concentration of trace elements in view of maximising the level of B, TC, β-CAR, γ-CAR, TN and TD. After optimization, average final values total carotenoids (TC = 803.2 μg/g DW) was about 370% of value observed as central point of the central composite design scheme. Under the same condition, average final values of other responses were: B = 5.40 g/L; β-CAR = 50.3%; γ-CAR = 15.4%; TN = 22.7%; TD = 11.6%. All above experimental data are in good agreement with calculated ones, thus confirming the reliability of the proposed empirical model in describing carotenoid production by R. graminis as a function of trace element concentrations.     

Effect of biomass pre-treatment and solvent extraction on beta-carotene and lycopene recovery from Blakeslea trispora cells

The production of carotenoids from Blakeslea trispora cells in a synthetic medium has been reported, with the main products being beta-carotene, lycopene, and gamma-carotene. The effect of biomass pretreatment and solvent extraction on their selective recovery is reported here. Eight solvents of class II and III of the International Conference of Harmonization: ethanol, methanol, acetone, 2-propanol, pentane, hexane, ethyl acetate, and ethyl ether, and HPLC analysis were used for the evaluation of their selectivities towards the three main carotenoids with regard to different biomass pre-treatment. The average C(max) values (maximum concentration of caronoids in a specific solvent) were estimated to 16 mg/L with the five out of eight solvents investigated, whereas methanol, pentane, and hexane gave lower values of 10, 11, and 9 mg/L, respectively. The highest carotenoid yield was obtained in the case of wet biomass, where 44-56% is recovered with one solvent and three extractions and the rest is recovered only after subsequent treatment with acetone; thus, four extractions of 2.5 h are needed. Two extractions of 54 min are enough to recover carotenoids from dehydrated biomass, with the disadvantage of a high degree of degradation. Our results showed that, for maximum carotenoid recovery, ethyl ether, 2-propanol, and ethanol could be successfully used with biomass without prior treatment, whereas fractions enriched in beta-carotene or lycopene can be obtained by extraction with the proper solvent, thus avoiding degradation due to time-consuming processes.     

Single-cell oil production by Mortierella isabellina DSM 1414 using different sugars as carbon source

Pure carbon sources, especially carbohydrates which are raw materials deriving from agro-industrial processes, are often used for small-scale single-cell oil production by fermentation. The aim of this study was to investigate the effects of different pure carbon sources on cell growth, lipid accumulation, and γ-linolenic acid (GLA) production by the filamentous fungus Mortierella isabellina DSM 1414 (Deutsche Sammlung von Mikroorganismen). The sugars utilized in this study are found extensively and abundantly in nature, especially in food raw materials and, in consequence, in agro-food industry wastes or surpluses. Thus, the potential of many waste materials containing these sugars to be used in the production of single-cell oil by fermentation could also be evaluated. The effects of the sugars utilized on cell growth, biomass production, and lipid production were investigated. Fatty acids were also analysed in the lipids produced at the end of the fermentations. Results showed that the maximum biomass production was 10.80 g/L in lactose-based media, while the maximum oil production was 5.44 g/L in maltose-based media. Oleic (20.42%–42.94%), palmitic (14.96%–22.19%), and stearic (9.00%–26.92%) acids were the major fatty acids along with linoleic acid (11.35%–18.67%) and GLA (3.56%–8.04%). The production of GLA as the target fatty acid was remarkable. This study indicates that agro-industrial waste including most of the sugars utilized (except for arabinose and sucrose with lipid production of 0.81 and 0.28 g/L, respectively) can be employed for production of single-cell oil by M. isabellina DSM 1414 which contains a high amount of GLA.     

An alternative, banana peel-based medium used to investigate the catalytic properties of peroxidase from a fungus, Inonotus sp SP2, recently isolated in southern Chile

Recycling industrial wastes is one of the major goals of bioengineering research. Agricultural wastes are often rich in natural sources of organic and inorganic compounds. The present study investigates the use of banana peel waste as a non-conventional alternative to nitrogen-enriched glucose media for a white rot fungus (WRF), Inonotus sp. SP2, recently isolated in southern Chile. WRF are known to produce biodegrading enzymes, such as peroxidases, that can have industrial and biotechnological applications. To that end, the metabolic characteristics and catalytic properties of peroxidases produced by Inonotus sp. SP2 were compared between glucose and banana peel-based growth mediums. The results establish that this strain of WRF produces high concentrations of a Mn⁺²-dependent peroxidase, with greater enzymatic activity in extracellular fluid and crude enzyme extracted from fungus grown in banana peel and glucose media, respectively. H₂O₂ has an inhibiting effect that is greater for enzymes produced in glucose media, and greater biomass can be obtained in banana-peel based media. This demonstrates that banana peel is a suitable and more cost-effective alternative to conventional glucose-based media for the production of biodegradative enzymes, such as peroxidase. Unlike other strains of WRF, the metabolic characteristics of Inonotus sp. SP2 demonstrate that it enters secondary metabolism with the production oxidative enzymes after both carbon and nitrogen sources are depleted. This suggests that with further investigation, this strain of WRF may be useful in industrial applications that require the biodegradation of nitrogen and carbon-based wastes and recalcitrant compounds.