Induction of carotenoid pigments in callus cultures of Calendula officinalis L. in response to nitrogen and sucrose levels

In vitro carotenoid pigment production in callus cultures of Calendula officinalis L. was investigated using two basal media, semi-solid versus liquid media and varied concentrations of sucrose, ammonium, and nitrate nitrogen. Of the two explants that were evaluated, floret explants were best for callus induction using Murashige and Skoog (MS) medium supplemented with 2.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid under complete darkness. Carotenoid pigment induction was significantly augmented when the sucrose concentration was increased. Low sucrose concentrations in the culture medium deferred the onset of pigment induction and reduced the overall levels of carotenoid pigments produced. The highest amount of carotenoid pigments was observed when the callus was grown on the MS medium without ammonium nitrogen. The quantity of carotenoids was slightly elevated in cultures grown on semi-solid medium than those grown in liquid medium. In vitro carotenoid production was optimized by modifying the concentration of ammonium nitrogen to nitrate nitrogen in the culture medium and enhancing the sucrose concentration.     

Production of pigments byMonascus purpureus using sugar-cane bagasse in roller bottle cultures

Solid-state fermentation, using sugar-cane bagasse, and submerged fermentation, using a semi-synthetic medium, were performed for pigment production byMonascus purpureus in both stationary and rotary conditions. Rotary cultures gave higher yields of crude red and yellow pigments than stationary cultures whereas twice the amount was synthesized at an earlier time (day 8) in liquid medium (1,285U yellow pigment/bottle, 1,728U red pigment/bottle). Supplementing the liquid medium with 0.6% (v/v) corn oil doubled the extracellular pigment yield but halved fungal growth.     

The effect of different nitrogen sources on pigment production and sporulation of Monascus species in submerged, shaken culture.

Thirty-nine strains from the genus Monascus were cultivated aerobically to study the relation between nitrogen nutrition and sporulation and pigment production. The effects of yeast extract, nitrate, ammonium, and ammonium nitrate have been compared. During cultivation the pHs of the different media are not the same, resulting in the formation of different coloured pigments. When the source of nitrogen is yeast extract or nitrate the pH is around 6.5 and red pigments are formed, whereas with ammonium or ammonium nitrate the pH is around 2.5 and the pigments are orange. It is proposed that only the orange pigments, monascorubrin and rubropunctatin, are produced biosynthetically and that the other pigments are formed from these by chemical transformations depending on the cultural conditions. The presence of organic nitrogen is optimal for growth and unfavourable for pigment production. Reduced growth and best pigment formation occurs with the three other nitrogen sources. Nitrate stimulates conidiation and sexual reproduction, while ammonium is inhibitory. Pigment production is better when conidiation is reduced. A mechanism is proposed for the control of sporulation and pigment production.     

Effect of nutrition of Monascus sp. on formation of red pigments

Summary A higher producer of ascospores and pigments, Monascus strain TTWMB 6042, was used to study regulation of pigment production by nutrients. An initial medium containing 4% glucose, 0.3% NH₄NO₃ (75 mm nitrogen) and inorganic salts was used. We found that the formation of red pigments in this strain, measured by optical density at 500 nm (OD₅₀₀) was strongly stimulated by monosodium glutamate (MSG) as the sole nitrogen source. The choice of carbon source and an initial pH of pH 5.5 were also important. High concentrations of phosphate and MgSO₄ were inhibitory to pigment production. A new chemically defined medium was devised containing 5% maltose, 75 mm MSG, phosphate and MgSO₄ at lower concentrations plus other mineral salts, which yielded a tenfold increase in OD₅₀₀ and a reversal of the pigment location from predominantly cell-bound, including both intracellular and surface-bound pigments, to mainly extracellular. Offsprint requests to: A. L. Demain     

Production of pigments byMonascus purpureus in solid culture

Summary The effect of physical and nutritional factors on the production of pigments byMonascus purpureus FRR 2190 was studied using cultures grown on both rice and a synthetic medium that was solidified with carrageenan and extruded into rice-like particles. Pigment yield was highly sensitive to physical parameters. Optimal pigment formation in rice cultures occurred at an initial pH of 6 and an initial moisture content of 56%. Lower moisture contents led to a large decrease in pigment concentration. Red and yellow pigment production on solidified gel media was increased up to three-fold compared to that of liquid cultures of the same medium composition, particularly when peptone was used as the sole nitrogen source. Solid state rice cultures gave the highest pigment yields.     

Negative effect of ammonium nitrate as nitrogen source on the production of water-soluble red pigments by Monascus sp.

Ammonium salts, especially ammonium nitrate, have been used as nitrogen sources for production of traditional water-insoluble Monascus pigments. However, we noted that defined media employing NH₄NO₃ as the sole nitrogen source in fermentations supported only poor pigment production by Monascus sp., and the pigments produced were mainly cell-bound. NH₄NO₃ was found not to (a) repress pigment synthase formation, (b) enhance synthase decay, or (c) serve as a nitrogen source for pigment production by resting cells; it had a weak inhibitory effect on the action of pigment synthase(s). The high level of cell-bound did not exert a feedback effect on the further synthesis of pigments. These observations indicate that the reason why NH₄NO₃ supports only low pigment production during fermentations is the poor ability of NH₄NO₃ to donate nitrogen in the Schiff-base reaction converting orange pigments to red ones.     

AMMONIA PRODUCTION IN UREA-GROWN CULTURES OF CHLORELLA ELLIPSOIDEA12

Production of ammonia by urea-grown Chlorella ellipsoidea was investigated. Ammonia was produced during the stationary growth phase in cultures with urea as sole nitrogen source and glucose as supplementary carbon source. Ammonia was produced only in medium containing excess urea and limiting amounts of glucose. Ammonia production was accompanied by increase in pH. In cultures with nitrate as sole nitrogen source and glucose as supplementary carbon source, growth and pH changes were similar to those in urea-glucose medium, but no ammonia was detected. Cultures grown in urea-acetate medium were similar to those grown in urea medium without additional organic carbon source. No ammonia was produced under these circumstances and growth was significantly lower than that achieved in glucose-supplemented cultures. C. ellipsoidea evidently produces an enzyme or enzyme system which forms ammonia from urea. This organism was reportedly urease-free because previous workers did not detect ammonia formation from urea. Our findings indicate that special circumstances are required to produce detectable amounts of ammonia from urea. These findings are in agreement with a recent report of urea-splitting, cofactor-requiring enzyme in cell-free extracts of Chlorella.     

Enhancement of pigment productivity of immobilized cultured Lavandula vera cells by limitation of nitrogen sources

Cultured cells of Lavandula vera entrapped with a photosensitive synthetic resin prepolymer (PVA-SbQ) produced blue pigments in the presence of l-cysteine as an inducer. The type of nitrogen sources in the culture medium greatly influenced the production of pigments. In the absence of an ammonium type nitrogen source, the induction of pigment synthesis by l-cysteine was observed in successive batches of the incubation without intermittent activation of the cells in the absence of l-cysteine. The pigment productivity of the entrapped cells was remarkably enhanced in the improved production medium containing potassium nitrate as the sole nitrogen source.     

Interrelationships between carbohydrate metabolism and nitrogen assimilation in cultured plant cells

Summary The effect of the nature and concentration of the nitrogen source on respiratory activity and removal of carbohydrate from the medium in suspension cultured sycamore (Acer pseudoplatanus L.) cells was determined. Comparison was also made of the rates of uptake of the two alternative nitrogen sources, nitrate and glutamate, at differing initial nitrogen concentrations within the range 7–14 mM. The initial pH of the culture medium before inoculation was 5.2; after inoculation the pH of both nitrate and glutamate cultures rose to reach an eventual level in the range 7.0–7.1. Glutamate was removed from the medium more slowly than nitrate. Under the particular conditions of culture used the growth of the cells was nitrogen limited. Sugar uptake from the medium continued for some time after the nitrogen in the medium was depleted. The data show that although cell division and protein content are nitrogen-limited, dry weight and fresh weight yields may also be determined in a complex interaction through carbohydrate availability. There were no obvious differences in respiratory activity between cultures grown on nitrate or glutamate.     

A reduction in temperature induces bioactive red pigment production in a psychrotolerant Penicillium sp. GEU_37 isolated from Himalayan soil

Filamentous fungi are being globally explored for the production of industrially important bioactive compounds including pigments. In the present study, a cold and pH tolerant fungus strain Penicillium sp (GEU_37), isolated from the soil of Indian Himalaya, is characterized for the production of natural pigments as influenced by varying temperature conditions. The fungal strain produces a higher sporulation, exudation, and red diffusible pigment in Potato Dextrose (PD) at 15 °C as compared to 25 °C. In PD broth, a yellow pigment was observed at 25 °C. While measuring the effect of temperature and pH on red pigment production by GEU_37, 15 °C and pH 5, respectively, were observed to be the optimum conditions. Similarly, the effect of exogenous carbon and nitrogen sources and mineral salts on pigment production by GEU_37 was assessed in PD broth. However, no significant enhancement in pigmentation was observed. Chloroform extracted pigment was separated using thin layer chromatography (TLC) and column chromatography. The two separated fractions i.e., fractions I and II with Rf values 0.82 and 0.73, exhibited maximum light absorption, λ_max, at 360 nm and 510 nm, respectively. Characterization of pigments using GC–MS showed the presence of the compounds such as phenol, 2,4-bis (1,1-dimethylethyl) and eicosene from fraction I and derivatives of coumarine, friedooleanan, and stigmasterole in fraction II. However, LC-MS analysis detected the presence of derivatives of compound carotenoids from fraction II as well as derivative of chromenone and hydroxyquinoline as major compounds from both the fractions along with other numerous important bioactive compounds. The production of such bioactive pigments under low temperature conditions suggest their strategic role in ecological resilience by the fungal strain and may have biotechnological applications.     

Effect of pH on citrinin and red pigments production by Monascus purpureus CCT3802

The production of red pigments and citrinin by Monascus purpureus CCT3802 was investigated in submerged batch cultures performed in two phases: in the first phase, cells were grown on glucose, at pH 4.5, 5.5 or 6.5; after glucose depletion, pH was adjusted, when necessary, to 4.5, 5.5, 6.5, 7.0, 8.0 or 8.5, for a production phase. The highest total red pigments absorbance of 11.3 U was 16 times greater than the lowest absorbance and was achieved with growth at pH 5.5, followed by production at pH 8.5, which causes an immediate reduction of the intra cellular red pigments from 75% to 17% of the total absorbance. The lowest citrinin concentration, 5.5 mg L⁻¹, was verified in the same culture while the highest concentration, 55 mg L⁻¹, was verified in cultures entirely carried out at pH 5.5. An alkaline medium, besides promoting intra cellular red pigments excretion, strongly represses citrinin synthesis.     

Production and release of anthraquinone pigments by hairy roots of madder (Rubia tinctorum L.) under improved culture conditions

Carbon and nitrogen sources in the medium were selected for the culture of madder hairy roots producing anthraquinone pigments. The growth and pigment formation of the hairy roots were significantly enhanced by using modified Murashige-Skoog (MS) medium containing fructose and nitrate as the sole carbon and nitrogen sources, respectively, compared with those obtained in conventional MS medium with sucrose. Repeated-batch culture of the hairy roots was carried out, with pigment release into the medium obtained by means of O₂ starvation treatment. In three pigment-release operations during 29-d culture, the total amount of released pigments was 21 mg/dm³, representing an average production rate of 0.72 mg/(dm³·d).     

Production of red pigments byMonascus purpureus in solid-state culture

To maximize and sustain the productivity ofMonascus pigments, various environmental and nutritional parameters, such as the initial moisture content, pH, inoculum size, sample size, and nutrient supplement, that influence pigment production were evaluated in solid-state cultures as follows: initial moisture content, 50%; pH, 6.0; inoculum size 1 x 10⁴ spore cells (grams of dry solid substrate)⁻¹; sample size, 300 g. All supplementary nutrients (carbon, nitrogen, and mineral sources) added has inhibitory effects on the cell growth and red pigment production. In open tray culture the maximum biomass yield and specific productivity of red pigments were 223 mg DCW (grams of initial dry substrate)⁻¹ and, 47.6 OD₅₀₀ (DCW grams)⁻¹ h⁻¹, respectively.     

pH-mediated release of betalains from transformed root cultures of Beta vulgaris L.

Brief exposure of Beta vulgaris root cultures to acidic medium resulted in release of betalain pigments while the capability for regrowth and continued pigment accumulation was retained. A 10-min exposure to pH 2 followed by return to standard growth medium (pH 5.5, 1.1 mM PO₄) resulted in release of 0.59 mg pigment/g dry weight over the subsequent 24-h period. The released pigment corresponds to 36.8% of the total pigments. Further improvement in culture productivity was achieved through phosphate limitation. Specific pigment productivity increased fivefold for cultures grown in phosphate-free medium as compared to cultures grown in control medium (1.1 mM PO₄). A maximum total pigment production of 25.2 mg/l was observed at an initial medium phosphate level 0.3 mM. When combined with phosphate limitation, low pH facilitated the release of 3.03 mg pigment/g dry weight, which corresponds to 50% of the total pigment. The permeabilized roots were capable of regrowth and continued pigment accumulation. A cytochemical assay for respiratory activity revealed that the basis of regrowth was lateral root initials that were unaffected during the acidic pH treatment. Received: 16 December 1997 / Received revision: 7 May 1998 / Accepted: 16 May 1998     

Isolation of two novel purple naphthoquinone pigments concomitant with the bioactive red bikaverin and derivates thereof produced by Fusarium oxysporum

Filamentous fungi have gained growing interest as sources of diverse pigmented secondary metabolites. Some specific polyketides from Ascomycetous species have demonstrated a wide range of industrial applications in food, cosmetic, textile, and in the design of pharmaceutical products. The formulation of recipes containing fungal polyketides has increased over recent years. Fusarium strains were proven useful to mankind in a variety of technologies. Nevertheless, there is still need of new isolates of Fusarium for use in emerging and already existing fields. In this article, we report the concomitant production of the bioactive red bikaverin along with two novel purple pigments by the phytopathogenic Fusarium oxysporum LCP531 strain isolated from soil. In literature, the production of purple pigment had only been described in cultures of Fusarium Fujikuroi, Fusarium verticillioides, and Fusarium graminearum. The production of these naphthoquinonic pigments, their distribution (either produced in mycelia or excreted in liquid medium) and their chemical profiles were investigated with respect to nutrient composition. The pigments were extracted by using a pressurized liquid extraction method, monitored by colorimetric analysis and characterized by HPLC-DAD chromatography. To our knowledge, this is the first report of these two novel wild-type purple naphtoquinones pigments along with bikaverin, where additionally, the culture conditions were put into perspective to optimize fermentation cultures and extraction process accordingly to the pigment/biomolecule desired. These colored naphthoquinones should be promising fungal functional compounds which could be expected to have a place of choice, along with other antibacterial, antifungal, antiviral, anticancer, and antineoplastic derivatives. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2738, 2019     

Production of red pigments byMonascus purpureus in submerged culture

For the purpose of mass producingMonascus red pigments optimum medium composition and environmental conditions were investigated in submerged flask cultures. The optimum carbon and nitrogen sources were determined to be 30 g/L of glucose and 1.5 g/L of monosodium glutamate (MSG). Of the three metals examined, Fe²⁺ showed the stronges stimulatory effect on pigment production and some stimulatory effect was also found in Mn²⁺. Optimum pH and agitation speed were determined to be 6.5 and 700 rpm, respectively. Under the optimum culture conditions batch fermentation showed that the maximum biomass yield and specific productivity of red pigments were 0.20 g DCW/g glucose and, 32.5 OD₅₀₀ g DCW⁻¹ h⁻¹, respectively.     

Leucine interference in the production of water-soluble red Monascus pigments

The formation of soluble Monascus red pigments is strongly positively and negatively regulated by different amino acids. Leucine, valine, lysine, and methionine had strong negative effects on pigment formation. Leucine supported poor pigment formation when used as sole nitrogen source in fermentations, yet it neither repressed pigment synthase(s) nor inhibited its action. The new pigments derived from the hydrophobic leucine were more hydrophilic than the conventional red pigments (lacking an amino acid side-chain) and were extracellularly produced. Therefore, the low level of red pigments produced when leucine was the nitrogen source was not due to feed-back regulation by cell-bound leucine pigments. The negative effect of leucine was caused by enhanced decay of pigment synthase(s). The enhanced decay was not due simply to de novo synthesis of a leucine-induced protease.Abbreviations mSG Monosodium glutamate - MOPS 3-(N-morpholine)propane sulfonic acid - DCW dry cell weight     

The effect of changes in pH on phosphate and potassium uptake by Monascus rubiginosus ATCC 16367 in submerged shaken culture.

Monascus rubiginosus ATCC 16367 was cultivated aerobically in media containing ammonium and nitrate as nitrogen source. The pH of the medium was adjusted at different times, the pH of the nitrate medium being lowered to the pH of the ammonium medium and the pH of the ammonium medium raised to that of the nitrate. More phosphate was taken up on the nitrate medium, but potassium uptake did not start until 24h. On the ammonium medium, both were taken up in parallel from the beginning, but the amount of phosphate taken up never reached the same level as on nitrate medium. When the pH was adjusted, the uptakes changed, especially on the ammonium medium where a great increase in phosphate uptake was observed. More conidia were formed on the nitrate medium and more pigment on the ammonium medium. When the pH of either media was adjusted, the development of conidia and pigment production changed to that of the other control medium where the pH evolved normally in the direction of the change, regardless of the source of nitrogen. The reasons for the development of conidia on nitrate medium or where the pH is high, and the production of pigment on ammonium medium or at low pH is discussed.     

Growth kinetics of biopigment production by Thai isolated <Emphasis Type="Italic">Monascus purpureus</Emphasis> in a stirred tank bioreactor

Monascus purpureus is a biopigment-producing fungi whose pigments can be used in many biotechnological and food industries. The growth kinetics of biopigment production were investigated in a liquid fermentation medium in a 5-l stirred tank bioreactor at 30°C, pH 7, for 8 days with 100 rpm agitation and 1.38 × 10⁵ N/m² aeration. Thai Monascus purpureus strains TISTR 3002, 3180, 3090 and 3385 were studied for color production, growth kinetics and productivity. Citrinin as a toxic metabolite was measured from the Monascus fermentation broth. The biopigment productions were detected from fermentation broth by scanning spectra of each strain produced. Results showed a mixture of yellow, orange and red pigments with absorption peaks of pigments occurring at different wavelengths for the four strains. It was found that for each pigment color, the color production from the strains increased in the order TISTR 3002, 3180, 3090, 3385 with 3385 production being approximately 10 times that of 3002. Similar results were found for growth kinetics and productivity. HPLC results showed that citrinin was not produced under the culture conditions of this study. The L*, a* and b* values of the CIELAB color system were also obtained for the yellow, orange and red pigments produced from the TISTR 3002, 3180, 3090 and 3385 strains. The colors of the pigments ranged from burnt umber to deep red.     

The effect of pH and amino acids on conidiation and pigment production of Monascus major ATCC 16362 and Monascus rubiginosus ATCC 16367 in submerged shaken culture.

Monascus major ATCC 16362 and Monascus rubiginosus ATCC 16367 were cultivated aerobically on media containing nitrate or ammonium as nitrogen source to which the following modifications were made: (1) pH adjusted to 2.5 before sterilization; (2) addition of yeast extract; (3) addition of amino acids in identical proportions and concentrations to those found in yeast extract; (4) adjustment of pH to 2.5 after addition of amino acids. The addition of amino acids in the form of yeast extract increased mycelium formation and reduced conidiation and pigment production. The addition of an amino acid mixture did not increase mycelium formation to the same extent as yeast extract but increased the number of conidia, while pigment production was reduced, especially when nitrate was the nitrogen source. As the amino acids are taken up after conidial formation has started, it would appear that it is not the amino acids themselves which are directly responsible for the induction of conidiation. The addition of amino acids inhibits nitrate and ammonium uptake suggesting the need for an early intracellular nitrogen limitation to induce conidiation. Lowering the pH inhibits the formation of conidia and increases pigment production; also the effect of amino acid addition is totally annulled. The pH of the medium is all important in regulating the formation of conidia and pigment production. The possible effects of the pH on the uptake of certain medium components is discussed, as well as their possible control of certain metabolic pathways which ultimately determines the availability of intermediates for conidiation and pigment production.