Establishment and Bioreactor Cultivation of Morinda Elliptica Cell Cultures for the Production of Anthraquinones

Morinda elliptica (Rubiaceae) cell suspension cultures were established in shake flask and bioreactor systems for the production of anthraquinones (AQ). To improve AQ productivity at shake flask level, manipulations of media components such as carbon, nitrogen, phosphate and myo-inositol; and cultural conditions such as incubation temperature, light intensity, culture and inoculum age, were made. At bioreactor level, the study was aimed at finding the best bioreactor operation with minimmn foaming and wall-growth problem. Several strategies such as mode of aeration, number of impellers, paddle orientation, antifoam addition and medium fonnulations, were applied. Murashige and Skoog's basal medium was found to be the best medium in enhancing both cell growth and AQ production. By manipUlation of sucrose concentration, hormone combination and concentration, culture age and inoculum age, the type of medium formulation used to grow inoculum, incubation temperature and light intensity, three types of media were formulated - maintenance medium (M), growth medium (G) and production medium (P). The toxic effects of nitrogen were shown not a result of the individual effect of nitrogen toxicity per se but of both individual and collective effects of NR₄⁺ and N0₃⁻ levels, in consonance with the level of sucrose and the medium formulation used. Reduction in pH for cultures grown in medium containing high concentration of NR₄⁺ was another contributing factor for ammonium toxicity. Phosphate had little influence on cell growth and AQ production though its absence could suppress growth completely. The phosphate toxicity could also occur depending on sucrose level and medium formulation. Myoinositol was not an absolute requirement in M elliptica cell suspension culture. The growth of cell suspension cultures of M elliptica in G and P media were sigmoidal. The AQ yields in P medium, of 2.9 and 4.5 gl⁻₁ with corresponding overall productivity of 0.14 and 0.21 gl⁻₁ dl⁻₁ , under illumination and in the dark, respectively, were among the highest amount of secondary metabolites and productivities by plant cell suspension cultures. The formation of AQ displayed a non-growth associated characteristic. High sucrose, glucose and fructose concentration over the period of two weeks in P medium was suggested to cause osmotic pressure on the cells which hindered rapid growth, leading to higher accumulation of AQ. With increasing culture age to 36 month-old, the doubling time was increased by 30% to 1.5 days; and 100% to l .6 days, in M and P medium, respectively. The maximum cell concentration in LP(36) was however 35% lower than LP(l8) while the AQ yield dropped sharply from 2.92 g l⁻₁ to a mere 0.55 g l⁻₁ . The spent medium was observed more yellowish in LP(36) indicating that AQ was no longer retained in the cell vacuole but released into the medium. The faster rate of sucrose hydrolysis and uptake rate of glucose and fructose in LP(36) may have reduced the osmotic pressure in the medium which allows rapid cell growth and diffusion of AQ

Enhanced anthraquinones production from adsorbent-treated Morinda elliptica cell suspension cultures in production medium strategy.

Continuous removal of anthraquinones (AQ) by Amberlite polymeric adsorbents (XAD-4, XAD-7 and XAD-16) through in situ adsorption in Morinda elliptica cell suspension cultures is studied for product recovery and improvement of the overall titre. Ethanol was the best eluting solvent for effective recovery of AQ from all adsorbents. Pre-treatment of XAD-4 with sodium acetate not only enhanced intracellular AQ, but also AQ release and subsequent recovery from the adsorbent. The addition of sodium acetate pretreated XAD-4 on day 18 for 6-day contact period, achieved comparable cell growth to control (41 g/L), but with 1.3-fold higher intracellular AQ (124 mg/g DW) and two-fold increase in extracellular AQ (14.3 mg/L). High amount of adsorbent and longer contact period for the cultures entering stationary growth phase, stimulated AQ release and recovery but at the expense of cell growth. With 5–8.3 g XAD-4 adsorbent per litre M. elliptica culture in production (P) medium, between 60 and 90% AQ was recovered from extracellular AQ after 24–26 days of culture period

Anthraquinones production, hydrogen peroxide level and antioxidant vitamins in Morinda elliptica cell suspension cultures from intermediary and production medium strategies

The effects of medium strategies [maintenance (M), intermediary (G), and production (P) medium] on cell growth, anthraquinone (AQ) production, hydrogen peroxide (H2O2) level, lipid peroxidation, and antioxidant vitamins in Morinda elliptica cell suspension cultures were investigated. These were compared with third-stage leaf and 1-month-old callus culture. With P medium strategy, cell growth at 49 g l–1, intracellular AQ content at 42 mg g–1 DW, and H2O2 level at 9 mol g–1 FW medium were the highest as compared to the others. However, the extent of lipid peroxidation at 40.4 nmol g–1 FW and total carotenoids at 13.3 mg g–1 FW for cultures in P medium were comparable to that in the leaf, which had registered sevenfold lower AQ and 2.2-fold lower H2O2 levels. Vitamin C content at 30–120 g g–1 FW in all culture systems was almost half the leaf content. On the other hand, vitamin E content was around 400–500 g g–1 FW in 7-day-old cultures from all medium strategies and reduced to 50–150 g g–1 FW on day 14 and 21; as compared to 60 g g–1 FW in callus and 200 g g–1 FW in the leaf. This study suggests that medium strategies and cell growth phase in cell culture could influence the competition between primary and secondary metabolism, oxidative stresses and antioxidative measures. When compared with the leaf metabolism, these activities are dynamic depending on the types and availability of antioxidants

Growth and anthraquinone production of Morinda elliptica cell suspension cultures in a stirred-tank bioreactor

The effects of medium strategy, number of impellers, aeration mode, and mode of operation on Morinda elliptica cell suspension cultures in a stirred-tank bioreactor are described. A lower number of impellers and continuous aeration contributed toward high cell growth rate, whereas a higher number of impellers reduced cell growth rate, although not anthraquinone yield. The semicontinuous mode could indirectly imitate the larger scale version of production medium strategy and improved anthraquinone production even with 0.012% (v/v) antifoam addition. Production medium promoted both growth (maximum dry cell weight of 24.6 g/L) and anthraquinone formation (maximum content of 19.5 mg/g of dry cell weight), without any necessity for antifoam addition. Cultures in production medium or with higher growth rate and anthraquinone production were less acidic than cultures in growth medium or with lower growth rate and anthraquinone production. Using the best operating variables, growth of M. elliptica cells (24.6 g/L) and anthraquinone yield (0.25 g/L) were 45% and 140%, respectively, lower than those using a shake flask culture after 12 days of cultivation

Strategies to overcome foaming and wall-growth during the cultivation of Morinda elliptica cell suspension culture in a stirred-tank bioreactor

Strategies to overcome foaming and wall-growth during the cultivation of Morinda elliptica (Rubiaceae) cell suspension cultures in a stirred-tank bioreactor are described. Of all the strategies applied, only bubble-free aeration was successful in eliminating foaming by 100%. Despite the foaming effect of around 40% in G medium strategy with 0.012% (v/v) antifoam, the maximum dry cell weight attained (19.2 g 1-1) and anthraquinone (AQ) content (4.0 mg g-1 DW) was nearly three times higher than that achieved in cultivation using 0.025% (v/v) antifoam. For continuous cell growth, the effect of inoculum age should also be considered when anti-foam is to be added. P medium strategy, without antifoam addition, not only promoted both growth (18 g 1-1) and AQ production (9.8 mg g-1 DW), but also resulted in lower foaming and wall-growth (below 30% level), and higher foaming reduction (30-40%)

Characterization of Toxic Metals in Tobacco, Tobacco Smoke, and Cigarette Ash from Selected Imported and Local Brands in Pakistan

In this study, concentrations of Cd, Ni, Pb, and Cr were determined in tobacco, tobacco smoke-condensate, and cigarette ash for selected brands used in Pakistan. Smoking apparatus was designed for metal extraction from cigarette smoke. Samples were digested through microwave digester and then analyzed by flame atomic absorption spectrophotometer (FAAS). Higher concentration of Ni was detected in imported brands than the counterparts in the local brands. Pb levels were however higher in local brands while significant concentration of Cd was observed in both brands. For Cr, the level in tobacco of local brands was higher than their emitted smoke, whereas imported brands showed higher level in smoke than in tobacco. The cigarette ash retained 65 to 75% of the metal and about 25 to 30% went into the body. While this study revealed the serious requirement to standardize the manufacturing of tobacco products, more importantly is the urgent need for stronger enforcements to put in place to alert the general population about the hazardous effects of cigarettes and the health risks associated with these toxic metals

Characterization of a molybdenum-reducing Acinetobacter baumannii strain Serdang 1 with the capacity to grow on phenol and acrylamide

Contamination of organic xenobiotic pollutants and heavy metals in a contaminated site allows the use of multiple bacterial degraders or bacteria with the ability to detoxify numerous toxicants at the same time. A previously isolated SDS- degrading bacterium, Acinetobacter baumannii strain Serdang 1 was shown to reduce molybdenum to molybdenum-blue. The bacterium works optimally at pH 6.5, the temperature range between 25 and 34°C with glucose serves as the best electron donor for molybdate reduction. This bacterium required additional concentration of phosphate at 5.0 mM and molybdate between 15 and 25 mM. The absorption spectrum of the molybdenum blue obtained is similar to the molybdenum blue from other earlier reported molybdate reducing bacteria, as it resembles a reduced phosphomolybdate closely. Ag(i), As(v), Pb(ii) and Cu(ii) inhibited molybdenum reduction by 57.3, 36.8, 27.7 and 10.9%, respectively, at 1 p.p.m. Acrylamide was efficiently shown to support molybdenum reduction at a lower efficiency than glucose. Phenol, acrylamide and propionamide could support the growth of this bacterium independently of molybdenum reduction. This bacterium capability to detoxify several toxicants is an important tool for bioremediation in the tropical region

Characterization and identification of newly isolated Acinetobacter baumannii strain Serdang 1 for phenol removal

A new indigenous bacterial strain from Malaysian soil contaminated with petroleum waste had been successfully isolated, characterized and identified for phenol removal. The gram negative bacteria showed 98% identity with Acinetobacter baumannii based on Biolog™ Identification System and the determination of a partial 16S ribosomal RNA (rRNA) sequence. The isolate clustered with species belonging to Acinetobacter clade in a 16S rDNA-based neighbour-joining phylogenetic tree

Antitumor promoting and actioxidant activities of anthraquinones isolated from the cell suspension culture of Morinda elliptica

Six anthraquinones (nordamnacanthal, alizarin-1-methyl ether, rubiadin, soranjidiol, lucidin-ω-methyl ether and morindone) isolated from the cell suspension culture of Morinda elliptica were assayed for antitumor promoting and antioxidant activities. All compounds exhibited strong antitumor promoting activity at the concentration of 2.0 μg/ml when assayed using the inhibition test of Epstein Barr Virus (EBV) activation on Raji cells. At the concentration of 0.4 μg/ml, only nordamnacanthal exhibited strong antitumor promoting activity with the inhibition rate and the cell viability of 75.0% and 75.8%, respectively, which was stronger than the reference compounds genistein and quercetin. In antioxidant assay using ferric thiocyanate (FTC) method, nordamnacanthal and morindone showed stronger antioxidant activity than α-tocopherol. However when the compounds were assayed for scavenging activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, only morindone was considered to be active as free radical scavenger with fifty percent inhibition concentration (IC50) of 40.6 μg/ml

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