Increased anthraquinone production in Galium vernum cell cultures induced by polymeric adsorbents

Anthraquinones produced by suspension cultures of Galium vernum are completely retained intracellularly. Surprisingly, in the presence of some polymeric adsorbents anthraquinones are partially released into the culture medium. The secretion and in situ removal stimulates anthraquinone production in cell cultures of Galium vernum. Best results were obtained with Wofatit ES and Amberlite XAD-2.Abbreviations DW dry weight - MS Murashige & Skoog[7]medium - NAA 1-naphthaleneacetic acid     

Effects of nutritional factors on the formation of Anthraquinones in callus cultures of Rheum ribes

Rheum ribes L. (Polygonaceae) is the source of one of the most important crude drugs in Asiatic regions .The medicinal character of rhubarb is due to its anthraquinone content . Different parts of sterile seedlings were cultured on MS medium to study the generation of callus. The explants were cultured with different ranges of plant growth regulators and the best range of plant growth regulators for generation of callus was IBA (1 mg l^–1) and BA (1 mg l^–1). The content of anthraquinones were determined by HPLC . The concentration of sucrose, vitamins and Myo-inositol and ratio of NO₃ to NH₄ in the medium was changed and growth rate and content of 2 anthraquinones was determined . The growth rate of callus declined with increased rate of secondary metabolites production. Myo-inositol 100 mg l^–1 in the medium increased anthraquinone content and in medium that had NO₃/NH₄:1/1 the maximum content of anthraquinone was obtained.     

Effect of chitosan elicitation and media components on the production of anthraquinone colorants in madder (Rubia akane Nakai) cell culture

To increase the production of anthraquinone colorants in madder (Rubia akane Nakai) cell culture, the effects of elicitation on the colorant production were investigated. Chitosan was the best biotic elicitor among nine plant derived and microbial derived polysaccharides. When elicited with 25 mg/L chitosan, the total production was increased approximately two times in a seven-day culture as compared to that in the unelicited cells. Anthraquinone production was increased in proportion to the contact period up to day 3. Maximum anthraquinone colorants were obtained with 3-day treatment of chitosan. During chitosan elicitation, the total production was increased 1.3 times in MS medium containing galactose as compared to that containing sucrose. The degree of deacetylation in chitosan and the use of growth regulator or addition of precursor did not affect the production of anthraquinone colorants. When madder cells were elicited at optimum condition, anthraquinone concentration and specific anthraquinone content increased 1.3 times (0.69 g/L) and 2.2 times (0.32 g/g DCW), respectively.     

A chemically-defined medium for production of compactin by Penicillium citrinum

A mutant strain of Penicillium citrinum grown in a chemically-defined production medium, yielded 145 mg compactin l^–1. The medium also facilitated spectrophotometric analysis of compactin. Addition of KH₂PO₄in the production medium did not increase the compactin production, while addition of a surfactant, Tween 80, increased compactin to 175 mg l^–1. Inoculation with 10⁷ spores ml^–1 and initial pH of 6.5–7 were the most suitable for compactin production.     

Establishment of cell suspension cultures of Morinda elliptica for the production of anthraquinones

Morinda elliptica (Rubiaceae) cell suspension cultures were established in shake flask system for the production of anthraquinones. The optimized medium formulation for cell growth and anthraquinone production is proposed. Murashige and Skoog's basal medium (MS) was found to be the best medium, used in combination with 0.5 mg l-1 NAA and 0.5 mg l-1 kinetin. At the range of sucrose concentration tested (3–8% w/v), 8% was the best in enhancing both cell growth and anthraquinone production. A strategy to formulate growth and production medium by manipulating culture age and inoculum age, the type of medium formulation used to grow inoculum, incubation temperature and light intensity was established. By using 18 month old culture and 7 day old inoculum at incubation temperature of 27 ± 3 °C, anthraquinone yield of 2.9 g l-1 and 4.5 g l-1, under illumination of 1200 lux and in the dark was obtained, respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

The stimulation of anthraquinone production by Cinchona ledgeriana cultures with polymeric adsorbents

Summary Anthraquinones produced by suspension cultures of Cinchona ledgeriana are released into the medium, which becomes saturated with products late in the growth cycle. When a high affinity polymeric adsorbent, such as the macro-reticular Amberlite XAD-7, is added to the culture the concentration of anthraquinone in the medium is maintained at a low level and production may be stimulated 15-fold, yielding up to 20 mg/1/day. More than 90% of the product is released from the cells. For maximal yields it is shown that both the amount of adsorbent used and the time after sub-culture at which it is added to the system are critical. The value of such a method for product recovery from immobilised cells is discussed.     

Production of biogas from water hyacinth (Eichhornia crassipes) (Mart) (Solms) in a two-stage bioreactor

A two-stage rumen-derived anaerobic digestion process was tested for the conversion of water hyacinth shoots and a mixture of the shoots with cowdung (7:3) into biogas. Under conditions similar to those of the rumen and loading rates (LR) in the range of 11.6–19.3g volatile solids (VS) l–1d–1 in the rumen reactor, the degradation efficiencies were 38% for the shoots and 43% for the mixture. The major fermentation products were volatile fatty acids (VFA) with a maximum yield of 7.92mmolg–1 VS digested, and biogas with a yield of 0.2lg–1 VS digested. The effect of varying LR, solid retention time (SRT) and dilution rates on the extent of degradation of the water hyacinth–cowdung mixture was examined. Overall conversion of the substrate was highest at the loading rate of 15.4gVS.l–1d–1. Varying the retention times between 60 and 120h had no effect on the degradation efficiency, but a decrease was observed at retention times below 60h. The overall performance of the reactor was depressed by changing the dilution rate from 0.5 to 0.34h–1. By applying a LR of 15.4VS. l–1d–1, a SRT of 90h and a dilution rate of 0.5h–1 in the rumen reactor, and connecting it to a methanogenic reactor of the upflow anaerobic sludge blanket type, 100% conversion efficiency of the VFA into biogas with a methane content of 80% was achieved. The average methane gas yield was 0.44lg–1 VS digested.     

Optimization of thermostable lipase production from a thermophilic Geobacillus sp. using Box-Behnken experimental design

Thermostable lipase production by Geobacillus thermoleovorans was optimized in shake-flask cultures using Box-Behnken experimental design. An empirical model was developed through response surface methodology to describe the relationship between tested variables (Tween 80, olive oil, temperature and pH) and enzyme activity. Maximum enzyme activity (495 U l^–1) was attained with Tween 80 at 5 g l^–1; olive oil at 60 g l^–1; 70 °C and pH 9. Experimental verification of the model showed a validation of 95%, which is more than 4-fold increase compared to the basal medium.     

Accumulation of anthraquinones in Morinda citrifolia cell suspensions

Cell suspensions of Morinda citrifolia were cultivated in a B5-medium containing 4% sucrose as the sole carbon source and 1 mg l^-1 naphthyl acetic acid (NAA) or 1 mg l^-1 2,4-dichloro-phenoxyacetic acid (2,4-D). Both auxins were able to support growth but only in the presence of NAA anthraquinone production was observed. 2,4-D inhibited the production in NAA cultures. Anthraquinone synthesis took place in the growth and the stationary phase and amounts of 0.2–0.4 mmol (about 100–200 mg) g^-1 dry weight could be reached.Under both growth conditions sucrose was hydrolyzed extracellularly by invertase. From the resulting monosaccharides, glucose was taken up preferentially and an appreciable uptake of fructose only took place when medium glucose was exhausted. Sugar uptake rates were similar when cells were grown in NAA and in 2,4-D medium but the intracellular sugar contents (expressed on a dry weight basis) differed considerably. The presence of sucrose, glucose and fructose was demonstrated under both growth conditions. The amounts of sucrose and glucose were much lower in the 2,4-D cells than in the NAA-cells especially during the growth phase. Fructose contents were low and comparable, while in NAA cells an unknown sugar (possibly the sugar moiety of the glycosylated anthraquinones) was observed especially at the end of the growth phase and in the stationary phase. The differences in sugar concentrations were even larger due to the lower water contents of the NAA cells.Respiration of 2,4-D cells was much higher than that of NAA cells during the growth phase. A sharp increase in sugar contents (mainly sucrose) occurred in the 2,4-D cells at the end of the growth phase and corresponded with the fall in respiratory activity.A possible correlation between the lack of production of anthraquinones in 2,4-D cells and a less efficient growth metabolism in these cells is discussed.Abbreviations AQ anthraquinones - 2,4-D 2,4-dichloro-phenoxy-acetic acid - DW dry weight - FW fresh weight - NAA naphthyl acetic acid - pCPO p-chloro-phenoxy-acetic acid     

Improved production of ginseng polysaccharide by adding conditioned medium to Panax notoginseng cell cultures

By adding 50% (v/v) filtered culture broth to fresh MS medium, the specific growth rate of Panax notoginseng was increased from 0.046 d–1 to 0.068 d–1, and the polysaccharide production and productivity reached 1.21 g l–1 and 61 mg/(ld), respectively, which were 1.3- and 2.3-fold of the control. Further supplementation of the conditioned medium with sucrose, ammonium, nitrate and phosphate gave a cell density of 13.7 g l–1 and a specific growth rate of 0.086 d–1. Polysaccharide production was 1.65 g l–1 and the productivity was 78 mg/(ld).     

High scleroglucan production by Sclerotium rolfsii: Influence of medium composition

Scleroglucan production by Sclerotium rolfsii was markedly affected by the C-source concentration, showing a highest value with 150 g sucrose l–1. Production was also influenced by the N-source, being considerably higher in media containing NO3– than in those containing NH4, which had a clear inhibitory effect. Once defined the optimum culture medium composition, the highest exopolysaccharide production (ca. 26 g scleroglucan l–1) was achieved after 72 h of fermentation at shake flask scale. High values of yield (Yp/c = 0.49), productivity (Pr = 0.365 g l–1 h–1) and specific productivity (Pr/x = 0.031 g (g biomass)–1 h–1) were observed, and productivity was 1.5 times further increased by scalling-up to fermenter scale. Addition of L-threonine, sunflower oil and ascorbic acid diminished exopolysaccharide production. © Rapid Science Ltd. 1998     

Molecular weight of cytochrome oxidase

The molecular weight of bovine heart mitochondrial cytochrome oxidase in 2% or 3% deoxycholate was determined by the sedimentation velocity method to be 228,000 daltons from an S_20,w=8.44×10^–13 sec, a D_20,w=3.21×10^–7 cm² sec^–1, and the reported by others. The S_20,w value was only slightly concentration-dependent. When the deoxycholate in our preparation was replaced with Tween 80 the S value increased to between 16 and 17. When one preparation in Tween 80 was allowed to stand at room temperature, the S value increased in successive determinations to reach 51.5 at the end of approximately 7 h. The minimum molecular weight of the enzyme as calculated from the heme content (determined from the absorbance at 603 nm) and total protein content (determined from total nitrogen) was 110,000. An amino acid analysis when related to the heme content yielded a minimum molecular weight of 85,000.Deceased.     

Permeabilization and in situ adsorption studies during growth and coumarin production in hairy root cultures of Cichorium intybus L

Effect of addition of a permeabilizing agent dimethyl sulfoxide (DMSO) and a solid adsorbent, XAD -7, on growth and coumarin production in hairy root cultures of C. intybus was studied. Continuous permeabilization of the hairy root cultures of C. intybus with DMSO has been shown to be an effective strategy for enhanced release of coumarins while preserving the root viability. DMSO at 0.2% (v/v) level showed the maximum growth and coumarin production but was less as compared to control on day 28. Treatment of cells with increasing concentrations of DMSO (0.3 - 0.6 % v/v) to hairy root cultures of C. intybus, showed an inverse relationship with growth and coumarin production. Growth and production of coumarins increased with 1% media filtrate (MF) of cultures of Phytopthora parasitica var. nicotiana treatment. It was observed that treatment with DMSO (0.2% v/v) and 1% MF of P. parasitica showed the better growth and coumarin production with an increased release of coumarins as compared to the control and other treatments. It was observed that treatment of hairy root cultures with XAD-7 resulted in lesser growth and coumarin production as compared to control during the culture period. Addition of XAD-7 along with 1% MF of P. parasitica showed enhanced growth, coumarin production and increased adsorption as compared to control and lone XAD-7 treatment. Combined addition of DMSO/XAD-7 with fungal elicitor showed synergistic response in terms of biomass and coumarin production. Excretion of coumarins in both the cases was dependent on the presence of DMSO/XAD-7. These results showed that continuous permeabilization of hairy root cultures of C. intybus by using DMSO at 0.2% (v/v) level coupled with 1% MF of P. parasitica maintained viability of tissues and produced coumarins at higher level.     

Modelling the adsorption kinetics of erythromycin onto neutral and anionic resins

In this study the selective adsorption method was chosen to enable the recovery of erythromycin. The following sorbents were tested: neutral resins (XAD-4, XAD-7 and XAD-16) and an anionic resin (IRA-410). A mathematical kinetic model for the adsorption of erythromycin against time, on XAD-4, XAD-7 and XAD-16 resins, is proposed. Both Freundlich and Langmuir models showed a good fit for the sorbents XAD-7 and IRA-410 resins. The highest adsorption efficiency was observed when synthetic neutral resin, XAD-7 and XAD-16, were used. The estimated affinity and concentration factors show that the neutral resins tested are adequate for the selective adsorption of erythromycin. The estimated values of enthalpy and free energy of adsorption, lower than 12 kJ mol^–1 and –2 kJ mol^–1, respectively, indicate that a physiosorption process occurred.     

Taxol production in suspension cultures of Taxus baccata

The response of Taxus baccata (PC2) to basic manipulations of culture conditions is described. Suspension cultures of Taxus baccata (PC2) were maintained at 25°C on a modified B5 medium with two-week transfers. Under these conditions, no taxol^® is formed. However, if the cells are left in the same medium for 7 or more additional days, taxol is produced and released (ca. 90%) into the extracellular medium. Levels as high as 13 mg 1^–1 extracellular taxol were achieved in shake flask cultures and taxol was the primary taxane formed representing between 50 and 80% of total taxane in the medium. The cells are sensitive to changes in culture conditions and cultures cycle through periods of high (13 mg 1^–1) and low (<0.1 mg 1^–1) levels of taxol production during extended culture. Picloram was the most effective of the auxins tested with respect to cell growth but it suppressed taxol production. Addition of fructose to moderately-productive cultures (ca. 4 mg 1^–1) improved taxol production, but cultures in a high producing state did not respond. Glucose suppressed taxane production. Two isoprenoids (geraniol and pinene) had a modest effect on taxol production when added to cultures at 10 mg 1^–1.®|Taxol is a registered trademark of Bristol Meyer Squibb for paclitaxel     

Production of volatile compounds by the edible fungus Rhizopus oryzae during solid state cultivation on tropical agro-industrial substrates

When Rhizopus oryzae was grown on medium containing cassava bagasse plus soybean meal (5:5 w/w), CO2 production was at its highest (200 ml.l–1) while highest volatile metabolite production was with amaranth grain as substrate (282.8 ml.l–1). In the headspace, ethanol was the most abundant compound (more than 80%). Acetaldehyde, 1-propanol, ethyl acetate, ethyl propionate and 3-methyl butanol were also present. CO2 and volatile metabolite productions reached their maxima around 20 h and 36 h, respectively. © Rapid Science Ltd. 1998     

The production of anthraquinones by cell suspension cultures of Cinchona ledgeriana

In a suspension culture of Cinchona ledgeriana a range of anthraquinones accumulate. Fifteen aglycone components of this fraction have been identified, six of which (anthragallol-1,2,3-trimethyl ether, 1,4,5-trihydroxy-2 methylanthraquinone, 5-hydroxy-2-methylanthraquinone, 1,5-dimethoxy-2,3-methylenedioxyanthraquinone, 2,4,6-trihydroxy-1,3-dimethoxyanthraquinone and 1,2,5,6-tetramethoxyanthraquinone) have not previously been found in this species. The time course of anthraquinone production follows neither the growth of the cells nor alkaloid synthesis The anthraquinones are exported from the cells, with about 80 % of the aglycones present normally being recovered from the medium. High levels in the medium are found to inhibit growth of the culture. The final level accumulated shown to be dependent on a number of nutritional factors, the best yield being obtained when cells are grown with the growth regulators IBA (0.5 mg/l) and ZR (0.1 mg/l) present. Effectors ofthe shikimic acid pathway such as tryptophan and glyphosate are found to inhibit anthraquinone accumulation.     

Influence of feeding precursors on tropane alkaloid production during an abiotic stress in Datura innoxia transformed roots

The effects of feeding tropane alkaloid precursors in transformed root culture of Datura innoxia Mill. were studied during a stress treatment. The permeabilizing effect of Tween 20 on tropane alkaloid production by hairy root cultures was studied in flasks with different feeding of precursors (L-ornithine, L-arginine, L-phenylalanine, DL-β-phenyllactic acid, and tropinone). It has been shown that the addition of various precursors alone (0.5 m mol l ^-1) was ineffective in stimulating hyoscyamine production. In contrast, a short treatment with Tween 20, combined with L-phenylalanine feeding, amplified the level of hyoscyamine released into the medium compared with the Tween treatment alone. Thus, the total hyoscyamine content per flask was increased (+ 40%) compared with the control. When DL-β-phenyllactic acid (0.5 m mol l ^-1) was used, this last effect became more pronounced (+ 60%). These results show that permeabilization with Tween modulates tropane alkaloid accumulation by a release of alkaloids into the medium and a restoration of hyoscyamine root content. The simultaneous feeding of DL-β-phenyllactic acid and tropinone during the Tween treatment gave a similar effect to that obtained with DL-β-phenyllactic acid and Tween, suggesting that the synthesis of the tropate moiety determines the flux at the level of the esterification of tropine. This revised version was published online in June 2006 with corrections to the Cover Date.     

Bioconversion of linoleic acid into conjugated linoleic acid during fermentation and by washed cells of Lactobacillus reuteri

Conjugated linoleic acid (CLA) was produced at 300 mg l^–1 after 24 h culture of Lactobacillus reuteri in de Man–Rogosa–Sharpe medium containing 0.9 g linoleic acid (LA) l^–1 and 1.67% (v/v) Tween 80. CLA was mainly located in the extracellular space of the cells. Washed cells previously grown on LA were less active than unadapted washed cells in converting LA into CLA. Most of the CLA transformed by washed L. reuteri cells was located in cells or associated with cells. CLA production by washed L. reuteri cells was most efficient in conversion with 0.45 g LA l^–1 at pH 9.5 and 37°C for 1 h.     

Production of Oenococcus oeni biomass to induce malolactic fermentation in wine by control of pH and substrate addition

To increase the commercial production of Oenococcus oeni strains to be used for biological deacidification of wines, substrates addition and pH control have been optimized. The highest biomass yield of Oenococcus oeni (Y=6.9 mg mmol–1 sugar) was obtained when 55 mmol glucose l–1 and 30 mmol fructose l–1 were added both to the culture medium, and the pH was controlled at 4.8. Fructose was used as carbon and energy source, but also as electron acceptor improving the ability to reoxidize NAD(P)H.