Jean Archambault

Nutritional and hormonal requirements of Ginkgo biloba embryo-derived callus and suspension cell culture.

Calli were obtained from Ginkgo biloba embryos grown on Murashige and Skoog (MS) medium. The G. biloba cells could grow on either MS or Gamborg B5 mineral salt medium supplemented with sucrose (3% and 2%, respectively) and naphthaleneacetic acid (NAA) and kinetin (K) in concentrations ranging from 0.1 to 2.0 mg·L−1. Best growth and maintenance of callus cultures were achieved using MS medium supplemented with 2 mg·L−1 NAA and 1 mg·L−1 K (N2K1MS). Light was required to maintain healthy growth of the callus tissue.In both MS and B5 based media, sucrose was hydrolyzed extracellularly before being taken up by Ginkgo cell suspension cultures. Specific growth rates of 0.13 d−1 and 0.08 d−1 were obtained in MS medium supplemented with 1 mg·L−1 NAA, 0.1 mg·L−1 K and 30 g·L−1 sucrose (N1K0.1MS) and B5 medium supplemented with the same growth regulator regime and 20 g·L−1 sucrose (N1K0.1B5) respectively. Complete phosphate and ammonium uptake was observed in 11 days when cultured in MS medium and 10 days and 4 days respectively when cultured in B5 medium. During the culture, G. biloba cells consumed only 64% and 29% of the nitrate content of N1K0.1MS and N1K0.1B5 media respectively. Maximum dry biomass concentrations were 13.4 g·L−1 and 7.9 g·L−1, and yields on carbohydrate were 0.39 and 0.45 in N1K0.1MS and N1K0.1B5 media respectively. The better performance of MS cultures came from the higher sucrose and nitrogen salts concentrations of this medium.

Gas chromatographic-mass spectrometric analysis of ginkgolides produced by Ginkgo biloba cell culture

An analytical method was developed to confirm the production of ginkgolides by Ginkgo biloba cells cultured in vitro. Biomass samples were extracted for 16 h with acetone. This extract was loaded onto a silica gel column and purified by differential elution using a cyclohexane-ethyl acetate solvent system. The recoveries of ginkgolide A and ginkgolide B were × ei90% and × ei35%, respectively. Subsequently, this purified extract was trimethylsilylated for gas chromatographic separation of ginkgolide A and ginkgolide B. Flame-ionization detection was not selective enough for identification of ginkgolides A and B in the extracts. The ginkgolides were detected by coupling the gas chromatograph to a high-resolution mass spectrometer operated in the selected-ion monitoring mode. The concentration of ginkgolides A and B in culture extracts was determined by gas chromatography-mass spectrometry using deuterotrimethylsilylated ginkgolides as internal standards. This technique permitted detection of ginkgolides A and B at concentrations as low as 10− pmol/μl of injected purified extract.

Production of sanguinarine by elicited plant cell culture I. Shake flask suspension cultures

Abstract Various Papaver somniferum cell lines species were generated and examined for sanguinarine production either by elicitation with chitin or endogenous production. Initial elicitation experiments gave variable results, with sanguinarine yields and concentrations of up to 4.5% of the biomass dry weight and 202 mg l −1 , respectively, much higher than endogenous levels of production (~ 1 % or ~ 90 mg l −1 ). A closer examination of the growth and major nutrient (carbohydrates, phosphate, nitrate and ammonia) uptake patterns of these cultures revealed interesting insights into this process. Extracellular phosphate concentration near depletion (~ 0.1 mM) was found to be a reliable marker for elicitation and production. Elicited cultures showed an absolute requirement for extracellular carbohydrates and ammonia during the production phase for both survival and maximum production. Highest sanguinarine levels obtained were yield of 5.0%, concentration of 385 mg l −1 , and productivities of 82 and 26 mg l −1 d −1 for the production phase and whole culture duration, respectively.

Detection of ginkgolide A in Ginkgo biloba cell cultures

Ginkgo biloba cells were cultured in two 500 mL shake flasks and in 2 L and 6 L immobilization bioreactors using MS medium supplemented with 1 mg.L−1 NAA, 0.1 mg.L−1 K and 30 g.L−1 sucrose. Specific growth rates were 0.06 d−1, 0.11 d−1 and 0.07 d−1 for the 2 L and 6 L bioreactors and shake flask cultures, respectively. Extracellular phosphate, nitrate, ammonium and carbohydrate uptake rates of the bio reactor cultures were approximately 17 to 39% slower than those of shake flask cultures. The specific oxygen uptake and carbon dioxide transfer rates of immobilized Ginkgo biloba cells ranged from 0.027 to 0.041 mmol O2.g−1.d.w.hr−1 (maximum uptake at 14 days) and 0.020 to 0.057 mmol CO2g. −1.d.w.hr−1 (maximum production at 14 days). Extracts from the biomass of the two immobilized and shake flask suspension cultures were analysed for ginkgolide A by GC-MS. Yields of 7, 17, 19 and 7 ng.g. −1d.w. of ginkgolide A were determined for shake flask 1, shake flask 2 and the 2 L and 6 L immobilized cultures, respectively. Traces of ginkgolide B were detected with the signal to noise ratio, however, being too low for positive confirmation of this last product.

Physiological aspects of surface-immobilized Catharanthus roseus cells

SummaryGrowth and alkaloid production of surface-immobilized C. roseus cells were studied in a 2-1 bioreactor. Media designed to maximize cell growth or alkaloid production were employed. Nitrate and carbohydrate consumption rates as well as growth rates and biomass yields of immobilized cultures were equal or somewhat lower than for cell suspension cultures. Respiration rate (O2 consumption and CO2 production rates) of immobilized C. roseus cell cultures was obtained by on-line analysis of inlet and outlet gas composition using a mass spectrometer. Respiration rate increased during the growth phase and decreased once the nitrogen or the carbon source was depleted from the medium. The respiration rate of immobilized C. roseus cells resembled rates reported in the literature for suspension cultures.

Stimulated indole alkaloid release from Catharanthus roseus immobilized cultures. Initial studies

Vacuolar sequestration of valuable secondary metabolites remains the major limitation to the use of immobilization technology for large scale plant-cell-based bioprocesses, which otherwise may be a more efficient culture system than suspension for this biomass. In this initial study, the release of indole alkaloids produced by immobilized Catharanthus roseus cells cultured in Zenks Alkaloid Production Medium was evaluated. Unstimulated alkaloid release in immobilized cultures reached levels of 10 to 50% of total production or 3 to 100% of known alkaloid content (30 to 4700 micrograms l-1), which was higher than that found for suspension cultures of the cell line used (10 to 25% of total production) without apparent cell lysis. Modifications of the medium pH value of immobilized cultures were explored in order to improve this release. Periodical additions of acid (HCl 0.1 N) or base (KOH 0.1 N) solutions (2% v/v) to different cultures resulted in rapid (less than 3 h) and transient variations in extracellular pH value from 5.5 to 4.3, and 5.8 to 8.5, respectively. In both cases, these variations provoked significant increase in total alkaloid (from approximately 5-10 mg l-1 to 15 mg l-1), ajmalicine (from 0 to approximately 0.29 mg l-1) and serpentine (from 0 to approximately 0.20 mg l-1) release, without apparent cell lysis or decrease in the culture viability. This product release was estimated to represent 100% of alkaloids produced.

Effect of culture process on alkaloid production by Catharanthus roseus cells: I. Suspension cultures

The processes for production of indole alkaloids in shake flask suspension cultures of Catharanthus roseus cells using Zenks alkaloid production medium (APM) were evaluated. The 1-stage process consisted of inoculating APM and incubating for 15 days. The 2-stage process involved 6 d of cultivation in growth medium followed by 15 d of incubation in APM. Growth, main nutrient consumption and alkaloid production were monitored. Both culture processes produced approximately 20 g dw per 1 biomass. However, 2-stage cultures yielded an inorganic nutrient richer and more active plant cell biomass, richer in inorganic nutrients, as indicated by higher (greater than 70%) nutrient availability and consumption. Total and individual indole alkaloid production were 10 times higher (740 mg l-1 and 25 to 4000 micrograms per g dw, respectively) for 2-stage than for 1-stage cultures. For both processes, highest alkaloid productivity coincided with complete extracellular consumption of major inorganic nutrients, especially nitrate, by the cells. Complete carbohydrate consumption in 2-stage cultures resulted in a 40% decline in production. Small but significant (approximately 10%) product release was observed for both culture regimes, which seemed not to be related to cell lysis.

Effect of culture process on alkaloid production by Catharanthus roseus cells: II. Immobilized cultures

Two processes for the production of indole alkaloids 2 l surface-immobilized bioreactor cultures of Catharanthus roseus cells using Zenks Alkaloid Production Medium (APM) were evaluated. The 1-stage process consisted of inoculating APM containing bioreactors and incubating for 15 d. The 2-stage process involved inoculating growth medium-containing bioreactors, growing the immobilized cultures for a certain period of time and subsequently replacing this medium with APM. The production stage which lasted for 15 d. High production in 2-stage cultures required the replacement of the growth regulator 2,4-dichlorophenoxyacetic acid by indole-3-acetic acid in the growth medium and a growth stage of 6 d (late exponential phase) before production initiation. Growth, main nutrient consumption and alkaloid production were monitored. Both culture regimes resulted in similar biomass production, dw (10-13 g l-1). The 2-stage cultures yielded biomass richer in organic nutrients (200-300%) and with higher respiratory activity (approximately 250%), indicated by their lower biomass-to-carbohydrate yields (31% and 26%), as compared to 1-stage cultures (41%). Two-stage cultures produced more known products (10 as compared to 6) at yields (5 to 4800 micrograms g-1) 3 to 5 times higher than 1-stage cultures. More alkaloids were alkaloids released in the medium of 2-stage cultures, under non-lysing conditions, (20 to 4700 micrograms l-1) than in 1-stage cultures (20 to 460 micrograms l-1). These results were compared to those obtained from shake flask cultures performed at the same time, with the same C. roseus cell line and under similar regimes and reported previously. Suspension and immobilized cultures performed according to the 1-stage regime showed similar total production. However, release of known alkaloids was 2 to 3 times higher in immobilized than in suspension cultures. Total alkaloid production of 2-stage suspension cultures was 3.8-fold higher than 2-stage immobilized cultures. Two stage immobilized cultures released 4 more known alkaloids than the 2-stage suspensions. Lower oxygen availability in the 2 l immobilized cultures may explain lower specific growth rates (0.15-0.22 d-1) and total alkaloid production levels, compared to 200 ml suspension cultures (0.2-0.4 d-1) reported in our previous paper.

Solid-phase extraction and fluorimetric detection of benzophenanthridine alkaloids from Sanguinaria canadensis cell cultures

Abstract A solid-phase extraction (SPE) method using Sep-Pak C 18 reversed-phase cartridges was developed for the prepurification of benzophenanthridine alkaloids produced by Sanguinaria canadensis cell cultures. Efficient recoveries (>90%) of chelirubine, sanguinarine and chelerythrine were obtained from synthetic solutions using the SPE method. Up to 20 mg of alkaloids were extracted from spiked biomass and culture-medium samples on a 400-mg adsorbent cartridge. This extraction method was more reproducible and as selective as a conventional liquid—liquid extraction technique. Fluorimetric detection was used for the identification and quantification of benzophenanthridine alkaloids after high-performance liquid chromatographic separation. This detection method was compared to conventional UV absorption at 280 nm. The detection limits of both detectors were in the 100-pg range for chelirubine and sanguinarine and in the 1-ng range for chelerythrine. The main advantage of fluorimetry over UV spectrometry was its improved selectivity for the benzophenanthridine alkaloids.

Growth characteristics of Sanguinaria canadensis L. Cell suspensions and immobilized cultures for production of benzophenanthridine alkaloids

SummarySanguinaria canadensis L. plants were harvested from a local forest and calli were initiated from leaf explants. The production of benzophenanthridine alkaloids (i.e. sanguinarine, sanguilutine, sanguirubine, chelerythrine, chelilutine and chelirubine) by S. canadensis cell grown in modified B5 and IM2 media was compared to the alkaloid content of rhizomes. Sanguinarine accounted for approximately 80% of the total alkaloid content of cultured cells (1.3%,g g−1) while sanguinarine and sanguirubine accounted for 70% of rhizome alkaloids (9.0%, g g−1). Sanguinarine, chelirubine and chererythrine were the only known alkaloids detected in cultured S. canadensis cells. Maximum alkaloid production of cultures performed using B5 medium, containing half the original nitrate concentration, was observed following extracellular nitrate and sugar depletion. The scale-up of this culture was successfully performed in a 2-1 immobilization bioreactor. The consumption of sugar and nitrate as well as the oxygen (OTR) and carbon dioxide (CTR) transfer rates of the immobilized cell culture were monitored for 15 days. The maximum sugar and nitrate consumption rates were 1.8 g l−1 per day and 2.3 mm per day respectively. The maximum OTR and CTR of the immobilized cell culture were 0.8 mmol O2 l−1 h−1 and 0.95 mmol CO2 l−1 h−1 respectively. The sanguinarine yield of this culture reached 1.0% based on biomass dry weight (g g−1 dw) by day 15.