Influence of cobalt and other microelements on the production of betalains and the growth of suspension cultures of Beta vulgaris

The effects of six microelements (Cu²⁺, Mn²⁺, Fe²⁺, Mo²⁺, Zn²⁺, Co²⁺) on the production of betalains and the growth of suspension cultures of Beta vulgaris were studied. The increase of Co²⁺ from 1–5 M resulted in a 60% increment on the production of betalains. A positive effect of this divalent ion was only accomplished when it was added at the beginning of the culture. This was related to a doubling in the specific betalains production rate compared to B5 control medium. No effects on cell growth and ratio of betacyanines to betaxanthines were observed. Mo²⁺, Fe²⁺ and Cu²⁺ presented a positive but less marked effect, while the increase of Mn²⁺ did not show effects on the production of betalains compared to B5 control medium.     

Relationship between type and age of the inoculum cultures and betalains biosynthesis by Beta vulgaris hairy root culture

From a study of the relationship between the type and age of the inocula, and the growth and biosynthesis of betalains in a Beta vulgaris hairy root culture, the best results were achieved with a 14 d inoculum grown in submerged culture giving 42 mg betalains (16 mg betacyanins and 26 betaxanthins) and 1.5 g dry biomass in 40 ml medium.     

Characterization of recombinant Beta vulgaris 4,5-DOPA-extradiol-dioxygenase active in the biosynthesis of betalains

Betalains are water-soluble pigments with high antiradical capacity which bestow bright colors to flowers, fruits and other parts of most plants of the order Caryophyllales. The formation of the structural unit of all betalains, betalamic acid from the precursor amino acid 4,5-dihydroxyphenylalanine is catalyzed by the enzyme 4,5-DOPA-extradiol-dioxygenase followed by intramolecular cyclization of the 4,5-secodopa intermediate. This paper describes the purification and the molecular and functional characterization of an active 4,5-DOPA-extradiol-dioxygenase from the best-known source of betalains—Beta vulgaris—after heterologous expression in Escherichia coli. The enzyme is a monomeric protein with a molecular mass of 32 kDa characterized by chromatography, electrophoresis and MALDI-TOF analysis. Enzyme kinetic properties are characterized in the production of betalamic acid, the structural, chromophoric and bioactive unit of plant pigment betalains.     

Betalain producing cell cultures ofBeta vulgaris L. var. bikores monogerm (red beet)

Summary The betalains are a class of natural pigments comprising the yellow betaxanthins and the violet betacyanins. Callus lines developed fromBeta vulgaris, L. var. bikores monogerm exhibited cell colors ranging from white/green (nonpigmented) through yellow, orange, red, and violet and were representative of all betalain pigments found in the whole plant. The betalains have gained particular interest from the food industry as potential natural alternatives to synthetic food colorants in use today. Red beet extracts (E162), which contain significant amounts of the betacyanins, are currently used in products such as yogurts and ice creams. We describe here the characteristics of culture growth and betalain production for cell suspensions derived from the orange (predominantly betaxanthin-producing) and violet (betacyanin producing) callus lines. The major factors affecting betalain biosynthesis in both cultured and whole plant tissues are reviewed. Presented in the Session-in-Depth Batch Production and Fermentation at the 1991 World Congress on Cell and Tissue Culture, Anaheim, California, June 16–29, 1991.     

Betalain production in plant in vitro systems

Betalains have been widely used as natural colorants for many centuries, but their attractiveness for use as colorants of foods (or drugs and cosmetics) has increased recently due to their reportedly high anti-oxidative, free radical scavenging activities and concerns about the use of various synthetic alternatives. The main commercial sources of betalains are powders and concentrates of red beet (Beta vulgaris) or cactus pear (Opuntia ficus-indica) extracts. However, in recent years the technical and commercial feasibility of various in vitro systems to produce them biotechnologically has been explored. These research activities have included assessments of novel approaches for cultivating plant cell or tissue cultures, and diverse bioreactor systems for increasing production levels of secondary metabolites. This paper reviews recent progress in plant in vitro systems for producing betalain pigments. In addition, the factors that could be manipulated, the bioreactor systems that could be used, and the strategies that could be applied to improve betalain production are discussed.     

Scaled-up biotechnological production of individual betalains in a microbial system

The recent interest in plant pigment betalains as bioactive compounds and chemopreventive agents has led to the search for a reliable and scalable process to obtain them. The cloning of the novel and efficient enzyme 4,5-DOPA-extradiol dioxygenase from Gluconacetobacter diazotrophicus in an expression vector, and the subsequent heterologous expression in Escherichia coli cultures has led to the start-up of a biotechnological production system of individual pigments. The aim of this study was to search for the optimal conditions for the production of betalamic acid in microbial factories and the scaled-up obtention of the derived pigments. Four different betaxanthins and two betacyanins were obtained after the addition of non-transformable amines and amino acids and their condensation with the betalamic acid produced by the dioxygenase. The scaled-up obtention and purification of betalains improved the yields of the previous methodologies reaching quantities by up to 150 mg of pure compounds.     

Integrated Recovery of Pigments Released from Red Beet Hairy Roots Exposed to Acidic Medium

Hairy root cultures of Beta vulgaris L grown in a bubble column reactor were permeabilised by exposure to B5 medium of pH 2.0. The roots released 39% of their total pigments on a 10 min exposure to B5 medium of pH 2.0 followed by return to standard 135 medium. The pigments released in the extracellular medium were recovered on an adsorption column containing XAD-16 resin. The permeabilised roots regrew and accumulated additional pigments. Comparison of this technique with the previously used techniques like oxygen starvation and temperature shock to permeabilise beet hairy roots suggest that pH mediated release of betalains can be an effective method of releasing betalains from root cultures.     

Chemical composition of the cell wall in some green algae species

Cell walls of two strains ofChlorella vulgaris (from fresh water, F, and saline water, S) and ofKirchneriella lunaris contained various proportion of saccharides and proteins (highest percentage in dry matter was found inK. lunaris). They differed also in presence of individual monosaccharides (5 inC. vulgaris F, 8 inC. vulgaris S and 6 inK. lunaris) and amino acids (11 in C.vulgaris F, 6 in C.vulgaris S and 7 inK. lunaris). Common substances were rhamnose, cystine, proline, glutamic acid and leucine. Communicated by Z. ŠESTáK     

Variation in the phenology and abundance of flowering by native and exotic plants in subalpine meadows

The timing and abundance of flower production is important to the reproductive success of angiosperms as well as pollinators and floral and seed herbivores. Exotic plants often compete with native plants for space and limiting resources, potentially altering community floral dynamics. We used observations and a biomass-removal experiment to explore the effects of an invasive exotic flowering plant, Linaria vulgaris, on community and individual species flowering phenology and abundance in subalpine meadows in Colorado, USA. Invasion by L. vulgaris was associated with a shift in both the timing and abundance of community flowering. Invaded plant communities exhibited depressed flowering by 67% early in the season relative to uninvaded communities, but invaded sites produced 7.6 times more flowers than uninvaded sites once L. vulgaris began flowering. This increase in flowers at the end of the season was driven primarily by prolific flowering of L. vulgaris. We also found lower richness and evenness of resident flowering species in invaded plots during the period of L. vulgaris flowering. At the species level, a common native species (Potentilla pulcherrima) produced 71% fewer flowers in invaded relative to uninvaded plots, and the species had reduced duration of flowering in invaded relative to uninvaded sites. This result suggests that L. vulgaris does not simply alter the flowering of subordinate species but also the flowering of an individual common species in the plant community. We then used observational data to explore the relationship between L. vulgaris density and resident floral production but found only partial evidence that higher densities of L. vulgaris were associated with stronger effects on resident floral production. Taken together, results suggest that a dominant invasive plant can affect community and individual-species flowering.     

Qualitative and quantitative aspects of betalains biosynthesis in Amaranthus caudatus L. var. pendula seedlings

Seedlings of Amaranthus caudatus L. var. Pendula were used to study the influence of several treatment: white light, 3,4-dihydroxyphenylalanine (DOPA), kinetin, gibberellic acid (GA₃) on betalains biosynthesis. The pigments, betacyanins and betaxanthins, were separated using a Sephadex G-15 column chromatography. Qualitative as well as quantitative differences were observed according to the treatments applied.The amaranthin biosynthesis seemed to be favored in the absence of DOPA. Under the combined effect of kinetin and white light a small quantity of betanin was also synthesized. Adding exogenous DOPA led to a more diversified production which included betacyanins (amaranthin and betanin), betaxanthins (vulgaxanthin and miraxanthin), and even dopachrome. As a general rule, kinetin activated the betalains biosynthesis whereas GA₃ inhibited it. The stimulating effect of white light was always much greater than that of kinetin.Abbreviations GA₃ gibberellic acid - GA₄ gibberellin 4 - BHT 2,6-diter-butyl-4-methylphenol - DOPA 3,4-dihydroxyphenylalanine - Kinetin 6-furfurylaminopurine     

Verbascoside production by plant cell cultures

Verbascoside was found to be produced in all calli derived from eleven species that contained the compound in their leaves. Cell suspension cultures were also established in three species, i.e., Leucosceptrum japonicum f. barbinerve, Syringa josikaea, and Sy. vulgaris, all of which were found to produce verbascoside at more than 1 g/l. Of the three species, suspension cultures of L. japonicum f. barbinerve showed rapid growth and the highest yield of verbascoside (1.89 g/l). In these cultures, the effects of major salt concentration in B5 medium on cell growth and verbascoside production were examined. Maximum cell growth and maximum verbascoside production were both achieved by reducing the major salt concentration to half that of the original medium.     

Role of microalgal ligninolytic enzymes in industrial dye decolorization

Abstract

In this study, the decolorization efficiency of seven microalgae isolates; Nostoc muscorum, Nostoc humifusum, Spirulina platensis, Anabaena oryzae, Wollea saccata, Oscillatoria sp. and Chlorella vulgaris was investigated for dye decolorization. The highest decolorization percentages of Brazilwood, Orange G, and Naphthol Green B dyes (99.5%, 99.5%, and 98.5%, respectively) were achieved by Chlorella vulgaris. However, the maximum efficiency for dye decolorization percentages of CV and malachite green dyes were exhibited by A. oryzae (97.4%) and W. saccata (93.3%). Ligninolytic enzymes activity assay was carried out for laccase and lignin peroxidase enzymes, which revealed a high efficiency of the C. vulgaris, A. oryzae and W. saccata to lignin containing compound degradation. The highest laccase production recorded by C. vulgaris with Brazilwood, Orange G, and Naphthol Green B dyes (665.0, 678.6, and 659.5?U/ml, respectively). Similarly, C. vulgaris gave a high lignin peroxidase enzyme production with the above three dyes respectively (306.00, 298.34, and 311.45?U/ml). In addition, A. oryzae and W. saccata showed the highest production of the laccase enzyme (634.6 and 577.45?U/ml, respectively) with CV and malachite green dyes. The degradation products have been characterized after decolorization and verified using FTIR analysis. The high decolorization percentages achieved by C. vulgaris, A. oryzae and W. saccata make them potential candidates for bioremediation and pre-processing to remove dyes from textile effluents.     

Statistical evaluation and modeling of cheap substrate-based cultivation medium of Chlorella vulgaris to enhance microalgae lipid as new potential feedstock for biolubricant

Chlorella vulgaris (C. vulgaris) microalga was investigated as a new potential feedstock for the production of biodegradable lubricant. In order to enhance microalgae lipid for biolubricant production, mixotrophic growth of C. vulgaris was optimized using statistical analysis of Plackett–Burman (P-B) and response surface methodology (RSM). A cheap substrate-based medium of molasses and corn steep liquor (CSL) was used instead of expensive mineral salts to reduce the total cost of microalgae production. The effects of molasses and CSL concentration (cheap substrates) and light intensity on the growth of microalgae and their lipid content were analyzed and modeled. Designed models by RSM showed good compatibility with a 95% confidence level when compared to the cultivation system. According to the models, optimal cultivation conditions were obtained with biomass productivity of 0.123 g L^?1 day^?1 and lipid dry weight of 0.64 g L^?1 as 35% of dry weight of C. vulgaris. The extracted microalgae lipid presented useful fatty acid for biolubricant production with viscosities of 42.00 cSt at 40°C and 8.500 cSt at 100°C, viscosity index of 185, flash point of 185°C, and pour point of ?6°C. These properties showed that microalgae lipid could be used as potential feedstock for biolubricant production.     

Cell surface properties as factors involved in Proteus vulgaris adhesion to stainless steel under starvation conditions

The purpose of these investigations was to evaluate the influence of limited nutrient availability in the culture medium on Proteus vulgaris biofilm formation on surfaces of stainless steel. The relationship between the P. vulgaris adhesion to the abiotic surfaces, the cellular ATP levels, cell surface hydrophobicity and changes in the profiles of extracellular proteins and lipopolysaccharides was examined. In all experimental variants the starvation conditions induced the bacterial cells to adhere to the surfaces of stainless steel. Higher ATP content and lower cell surface hydrophobicity of P. vulgaris cells was observed upon nutrient-limited conditions. Under starvation conditions a reduction in the levels of extracellular low molecular weight proteins was noticed. High molecular weight proteins formed the conditioning layer on stainless steel plates, making the bacteria adhesion process more favorable. The production of low molecular weight carbohydrates promoted more advanced stages of P. vulgaris biofilm formation process on the surfaces of stainless steel upon starvation.     

Production of superoxide dismutases from Proteus mirabilis and Proteus vulgaris

Proteus mirabilis and Proteus vulgaris expressed a combination of superoxide dismutase (Sod) activities, which was assigned to FeSod1, FeSod2 and MnSod for P. mirabilis, and FeSod, MnSod and CuZnSod for P. vulgaris. Production of the Sod proteins was dependent on the availability of iron, whether cells were grown under anaerobiosis or aerobiosis and growth phase. Nalidixic acid and chloramphenicol inhibited cell growth and the iron- and dioxygen-dependent production of Sod. These results support the involvement of metal ions and redox status in the production of Proteus Sods.     

Genetic evidence for the origin of Californian wild beets (genus Beta)

The genus Beta L. is a morphologically and genetically variable group composed of wild, weedy, and domesticated forms that are used for sugar production or as vegetables. In this study, we have evaluated genetic variation in 64 germplasm accessions of wild and domesticated beets and examined the origin of wild beet accessions in California using allozyme analysis. UPGMA analysis showed overall that domesticated and wild beets form genetically coherent groups. Wild beets in California have two different origins, from European Beta vulgaris or from Beta macrocarpa. Population-level patterns of allozyme variation for wild California beets related to B. vulgaris suggest that those populations evolved from naturalized populations of the cultivated B. vulgaris ssp. vulgaris which had hybridized to varying degrees with the sea beets B. vulgaris ssp. maritima. Wild California beets related to B. macrocarpa are essentially genetically identical to European accessions. In addition, we found substantial evidence for hybridization and introgression of B. vulgaris alleles in one B. macrocarpa accession in California. The obligate outcrosser B. vulgaris exhibits more allelic diversity than the self-compatible B. macrocarpa. Beta vulgaris ssp. maritima exhibits more genetic diversity than domesticated B. vulgaris ssp. vulgaris. Received: 2 November 1998 / Accepted: 29 April 1999     

Short-term UV-B radiation effects on morphology,physiological traits and accumulation of bioactive compounds in Prunella vulgaris L.

Prunella vulgaris plants (full-bloom stage) were subjected to short-term (15 days) UV-B radiation in a growth chamber. UV-B radiation was effective at enhancing morphological and biomass characteristics and decreasing chlorophyll contents of P. vulgaris. Treatment of P. vulgaris with artificial UV-B radiation significantly increased peroxidase (POD), superoxide dismutase (SOD) and glutathione (GSH) activities compared to the control treatment. UV-B radiation significantly increased the levels of hydrogen peroxide (H₂O₂), malondialdehyde (MDA) and proline in leaves of P. vulgaris compared to those of control plants. In addition, the contents of total flavonoids, rosmarinic acid, caffeic acid and hyperoside significantly increased under UV-B radiation. The total phenolic levels also increased under UV-B treatment. These results demonstrated that short-term UV-B radiation can enhance production of secondary metabolites in P. vulgaris, resulting in increased spica yield compared to that of control plants.     

Thermophilic,anaerobic co-digestion of microalgal biomass and cellulose for H<Subscript>2</Subscript> production

Microalgal biomass has been a focus in the sustainable energy field, especially biodiesel production. The purpose of this study was to assess the feasibility of treating microalgal biomass and cellulose by anaerobic digestion for H₂ production. A microbial consortium, TC60, known to degrade cellulose and other plant polymers, was enriched on a mixture of cellulose and green microalgal biomass of Dunaliella tertiolecta, a marine species, or Chlorella vulgaris, a freshwater species. After five enrichment steps at 60°C, hydrogen yields increased at least 10% under all conditions. Anaerobic digestion of D. tertiolecta and cellulose by TC60 produced 7.7 mmol H₂/g volatile solids (VS) which were higher than the levels (2.9–4.2 mmol/g VS) obtained with cellulose and C. vulgaris biomass. Both microalgal slurries contained satellite prokaryotes. The C. vulgaris slurry, without TC60 inoculation, generated H₂ levels on par with that of TC60 on cellulose alone. The biomass-fed anaerobic digestion resulted in large shifts in short chain fatty acid concentrations and increased ammonium levels. Growth and H₂ production increased when TC60 was grown on a combination of D. tertiolecta and cellulose due to nutrients released from algal cells via lysis. The results indicated that satellite heterotrophs from C. vulgaris produced H₂ but the Chlorella biomass was not substantially degraded by TC60. To date, this is the first study to examine H₂ production by anaerobic digestion of microalgal biomass. The results indicate that H₂ production is feasible but higher yields could be achieved by optimization of the bioprocess conditions including biomass pretreatment.     

Influence of polyamines on growth and formation of secondary metabolites in hairy root cultures of Beta vulgaris and Tagetes patula

Growth of hairy roots of Beta vulgaris, which produces betalaines, and of Tagetes patula, which produces thiophenes, was studied under the influence of externally treated polyamines. Of the three polyamines, viz. putrescine, spermidine and spermine, administered singly at 1.5 mM concentration, putrescine and spermidine at 0.75 mM concentration influenced increase in the accumulation of biomass of B. vulgaris and T. patula hairy roots by 1.42 and 1.30 fold over the control. Whereas, the treatment of spermine (1.5 mM) alone resulted in decrease in the biomass in both the systems. Combined administration of putrescine (0.75 mM) and spermidine (0.75 mM) enhanced growth in both B. vulgaris and T. patula than that observed in individual treatments. Polyamines administered alone or in combination did alter production of betalaine and thiophene content. Dose response experiments showed that, when putrescine and spermidine was administered at 0.75 mM concentration, it resulted in maximum biomass and production of beta-laine and thiophene in B. vulgaris and T. patula respectively as compared to the control and the media treated with double and triple strength of nitrates and in combination with putrescine and spermidine at equimolar concentration. In B. vulgaris and T. patula hairy root cultures, endogenous spermine titers were maximum in putrescine and spermidine 0.75 mM each treated, cultures, which was 1.63 and 2.0 fold higher than in control on 28^th and 35^th days respectively.