Production of artemisinin by hairy rot cultures of Artemisia annua L in bioreactor

Hairy root cultures of Artemisia annua L were cultivated in four different culture systems: a flask, a bubble column, a modified bubble column and a modified inner-loop airlift bioreactor. The artemisinin contents of hairy root cultures in the bubble column and the modified inner-loop airlift bioreactor were higher than that in the modified bubble column. The growth rate and hairy root distribution in the modified inner-loop airlift bioreactor were better than those in other bioreactors, and dry weight and artemisinin production reached to 26.8 g/L and 536 mg/L after 20 days.     

Artemisinin Production by Adventitious Shoots of Artemisia annua in a Novel Mist Bioreactor

A novel ultrasonic inner-loop bioreactor was used for artemisinin production by adventitious shoots in a multiplate culture of Artemisia annua L. The bioreactor was designed to allow the nutrient mist to uprise along a concentric draught-cylinder until it overflows from the top opening and the side-holes of the central tube downward and out of the annulus, so that the nutrient mist can be fulfilled in the bioreactor within 2 ~ 3 minutes. Under the misting cycles of every 3-minute misting in every 90 minute interval, artemisinin production reached totally 46.9 mg DW/L of culture medium at an airflow rate of 0.5 L/min for 25 d of culture in batches. The product amounted 2.9 and 3.2 folds of those obtained from culturing in solid medium and in shaking flasks respectively.     

Growth of hairy root cultures of strawberry (Fragaria ¥ ananassa Duch.) in three different types of bioreactors

Hairy roots of strawberry were cultivated in three different types of bioreactors: an air-sparged bioreactor (control), a droplet bioreactor and a mist bioreactor. The highest biomass yields (3.7 g dry wt/l) were achieved in the air-sparged and in the mist bioreactor. In the droplet bioreactor the cultivation medium was insufficiently atomized into droplets and nutrient uptake and growth were slower due to uneven wetting of hairy roots.     

Performance of hairy root cultures of Cichorium intybus L. In bioreactors of different configurations

Summary A transformed root culture of Cichorium intybus L. cv. Lucknow Local grown in different configurations of bioreactors was examined. The roots grown in an acoustic mist bioreactor showed the best performance in terms of increased specific growth rate (0.072d⁻¹) and esculin content (18.5gl⁻¹), the latter of which was comparable to that of shake flask data. C. intybus hairy root cultures grown in an acoustic mist bioreactor produced nearly twice as much esculin as compared to roots grown in bubble column and nutrient sprinkle bioreactors. Studies relating to on-line estimation of conductivity and osmolarity to predict the growth of hairy root cultures are also discussed. The results demonstrate the efficacy and the advantages of an acoustic mist bioreactor for the cultivation of hairy root cultures, especially with reference to C. intybus hairy roots.     

Suitability of bench scale bioreactor system for shoot biomass production and bacoside biosynthesis from Bacopa monnieri (L.)

According to folklore, Bacopa monnieri commonly called as Brahmi is known for its cognitive enhancing properties. The plant is found abundantly in wetlands but the drug content (bacosides) is very low (0.2%), therefore, alternative biotechnological protocols are highly needed to supplement the constant source of this valuable plant material which produces stable amounts of bacosides. The present study was conducted to explore the application of different culture systems for cultivation of shoot biomass and maximization of biologically active bacoside biosynthesis in this medicinally important plant. Shoot cultures of Bacopa were cultivated in two different modified benchtop bioreactors: glass bottle bioreactor and balloon type bubble bioreactor and compared with those grown in traditional Erlenmeyer agitated flask. The shoots cultivated in the balloon type bubble bioreactor system showed excellent growth (growth index 796.47 ± 17.27 fresh weight and 395.55 ± 7.55 dry weight) as compared to glass bottle bioreactor system (growth index 488.17 ± 14.4 fresh weight and 327.79 ± 6.64 dry weight) and agitated flask (growth index 363.43 ± 11 fresh weight and 304.22 ± 6.76 dry weight). Furthermore, bacosides produced by shoot cultures cultivated in the balloon type bubble bioreactor (321.95 ± 17.14 mg/L) and glass bottle bioreactor (180.18 ± 6.25 mg/L) configurations were ～2.78 fold and ～1.55 fold higher than that recorded in agitated flask cultures (115.7 ± 3.84 mg/L). The balloon type bubble bioreactor system was found to be advantageous for enhancing B. monnieri shoot biomass and bacoside biosynthesis along with ensuring a successful protocol for continuous supply.     

Development of a nontoxic acoustic window nutrient-mist bioreactor and relevant growth data

Summary A nutrient-mist bioreactor was designed that separates the nutrient medium from the electronic components via an acoustic window. This eliminates compromising culture sterility when repairing mechanical failures common with commercially available mist reactors. The experimental mist bioreactor is low cost and can be assembled in any laboratory. Toxicity tests of several potential acoustically transparent materials are included. Details of the construction procedures include methods for casting the window. Growth data using the newly designed nutrient mist bioreactor are compared to data from a commercial mist reactor, shake flasks, and Gelrite cultures.Artemisia annua hairy roots andNephrolepis exaltata shoot cultures showed growth comparable to the conventional tissue culture methods.     

Multiple shoot culture of Dianthus caryophyllus using mist culture system

Summary A mist bioreactor system for the plant tissue cultures was developed. Using this system, the growth of Dianthus caryophyllus multiple shoots was directly measured. Tissue growth in mist bioreactor system was far better than that on agar medium and almost comparable to that in liquid medium. The mass increase (final dry weight/initial dry weight) in the mist culture was 2.85 while 3.28 in the liquid flask culture. Shoots were seriously vitrified in flask culture but these vitrifications could be considerably cured by using the mist culture system.     

Astragaloside IV and polysaccharide production by hairy roots of Astragalus membranaceus in bioreactors

Hairy roots of Astragalus membranaceus were grown in bioreactors up to 30 l for 20 d. Cultures from a 30 l airlift bioreactor gave 11.5 g l dry wt with 1.4 mg g^–1 astragaloside IV, similar to cultures from 250 ml and 1 l flasks, but greater than yields from a 10 l bioreactor (dry wt 9.4 g l^–1, astragaloside IV 0.9 mg g^–1). Polysaccharide yields were similar amongst the different bioreactors (range 25–32 mg g^–1). The active constituent content of the cells approached that of plant extracts, indicating that large scale hairy root cultures of A. membranaceus has the potential to provide an alternative to plant crops without compromising yield or pharmacological potential.     

Influence of mist intervals and aeration rate on growth and second metabolite production of Pseudostellaria heterophylla adventitious roots in a siphon-mist bioreactor

Plant adventitious root culture in bioreactors is a promising alternative for the efficient production of medicinal herbs. Adventitious roots of Pseudostellaria heterophylla were induced from callus and then cultivated in a siphon-mist bioreactor. An orthogonal test established that the optimal medium for adventitious root induction was MS medium supplemented with 1.0 mg/L naphthaleneacetic acid and 2.0 mg/L 3-indolybutyric acid. Under these conditions, the average root number was more than 14 on each 1.0 cm diameter callus and the rooting rate reached 100%. The bioreactor was equipped with an integral siphon-spraying device designed to automatically supply the liquid medium. The operation parameters of the bioreactor were assessed by varying the mist interval and the aeration velocity. The mist interval was negatively related to average growth rate of the adventitious roots and positively related to saponin and polysaccharide content. A relatively high aeration rate was necessary to achieve the maximum biomass production, but the secondary metabolite production was not enhanced by increasing the aeration velocity.     

Controlling hyperhydration of carnations (Dianthus caryophyllus L.) grown in a mist reactor

Carnations (Dianthus caryophyllus L.) grown in vitro often develop physiological abnormalities such as hyperhydration. The amount of hyperhydration and growth was compared between carnations grown in mist reactors and conventional semisolid micropropagation systems (vented or unvented GA7 culture boxes). Plants grown in the mist reactor with long misting times (10 min h(-1)) produced more dry mass than those grown with <10 min h(-1); however, more misting also produced more hyperhydrated plants (70% hyperhydration). Control of hyperhydration in the mist reactor involved either reducing the overall nutrient mist supply or altering the mist supply throughout the culturing period. Stepped decreases in the mist supply throughout the 3-week period or an overall decrease in the duration of misting reduced hyperhydration to 13% and 5%, respectively. However, for both misting regimes, the biomass of normal (healthy) plants (fresh and dry weights) was limited. Further analysis suggested that, although normal plant biomass increased with longer mist exposure, hyperhydration levels also increased while the water content, based on percent dry weight, approached that of hyperhydrated plants. Sufficient normal plant development (fresh weight, leaf and shoot numbers, height, and rooting) with < 50% hyperhydration was obtained by weekly, stepped increases in the nutrient mist supply.     

Methyl jasmonate effect on diterpenoid accumulation in Salvia sclarea hairy root culture in shake flasks and sprinkle bioreactor

Hairy root cultures of Salvia sclarea were grown in shake flasks and 10 L nutrient sprinkle bioreactor, running for 30 days and the effects of methyl jasmonate (MJ) on their growth and capacity to accumulate diterpenoids were measured. We found that MJ concentration and exposure time to the elicitor were factors that strongly affected the diterpenoid production. The highest diterpenoid accumulation (67.5 ± 7.1 mg g⁻¹ dry weight, calculated as a sum of ferruginol, salvipisone, aethiopinone and 1-oxoaethiopinone) without reduction of biomass, was achieved when the 23-day-old hairy roots in bioreactor culture were exposed to 125 μM MJ for 7 days. The roots produced 9 and 3.8 times as much aethiopinone (40 ± 5.9 mg g⁻¹ dry weight) and salvipisone (12.6 ± 0.4 mg g⁻¹ dry weight), respectively, as roots cultured in shake flasks. Our results imply that cultivation of S. sclarea hairy roots in sprinkle bioreactor after elicitation with MJ may be valuable to enhance production of the bioactive diterpenoids.     

Culture of amebocytes in a nutrient mist bioreactor

Summary We report the first use of nutrient mist bioreactor (NMB) technology to culture animal cells. The nutrient mist approximated the amebocyte stem tissue’s natural environment, which is a thin layer of fluid in the gill leaflets of the horseshoe crabLimulus polyphemus. NMB culture was tried in an attempt to increase production of amebocytes, which are the source of theLimulus Amebocyte Lysate (LAL), the basis for a sensitive and commercially valuable endotoxin assay. Amebocyte growth in the nutrient mist bioreactor is comparable to growth in liquid medium. However, the current design of the bioreactor presents problems for primary cultures such as ours where a pyrogen-free environment is necessary and fungal decontamination is difficult.     

Continuous production of human erythropoietin by immobilized recombinant L-929 cells

Recombinant L-929 cells transfected with the human erythropoietin (EPO) gene were immobilized in a macroporous cellulosic support and its derivatives in which charged groups or cell attachment factors were introduced. The immobilized cells were cultured in serum-containing and serum-free media. Comparable production of EPO was observed even in the serum-free medium when a support modified by polyethyleneimine was used for immobilization. The cells immobilized on the supports were cultured in fluidized-bed and inner-loop type air-lift bioreactors for continuous production of EPO. A high cell density of more than 2 × 10⁷ cells/cm³-support and high EPO productivity were achieved and maintained for 50 d through the use of the inner-loop type air-lift bioreactor. The productivity was 13.4-fold higher than that of conventional static cultures in petri-dishes.     

Growth of transformed roots in a nutrient mist bioreactor: reactor performance and evaluation

Summary A nutrient mist bioreactor was modified for culturing transformed roots of Beta vulgaris and Carthamus tinctorius on a nylon support. Culture conditions of misting cycle, inoculum size, batch or continuous operation and sucrose concentration were varied in order to maximize growth over a 1-week period. Root tissue cultured in nutrient mists in a 1.8-1 culture chamber achieved levels of growth equivalent to hairy roots cultured in shake flasks with identical medium. Our results demonstrate the effectiveness of nutrient mist culture as applied to hairy roots, thereby providing an alternative means for successful culture of these tissues. Correspondence to: A. A. DiIorio     

Bioreactor application on adventitious root culture of <Emphasis Type="Italic">Astragalus membranaceus</Emphasis>

Astragalus membranaceus is one of the most widely used traditional medicinal herbs in China, but the time required to generate a useful product in the field production is long. The growth of adventitious root cultures was compared between cultures grown in solid, liquid, or a 5-L balloon-type bubble bioreactor. The maximum growth ratio (final dry weight/initial dry weight) was determined for adventitious roots grown in the bioreactor. Studies carried out to optimize biomass production of adventitious roots compared adventitious root growth from various inoculum root lengths, inoculum densities, and aeration volume in the bioreactors. The maximum growth ratio occurred in treatments with a 1.5-cm inoculum root length, with 30 g (fresh weight) of inoculum per bioreactor or with an aeration volume of 0.1 vvm (air volume/culture medium volume per min). The polysaccharide, saponin, and flavonoid content of roots from bioreactor-grown cultures were compared to roots from field-grown plants grown for 1 and 3 yr. Total polysaccharide content of adventitious roots in the bioreactor (30.0 mg g⁻¹ dry weight (DW)) was higher than the roots of 1-yr-old (13.8 mg g⁻¹ DW) and 3-yr-old (21.1 mg g⁻¹ DW) plants in the field. Total saponin (3.4 mg g⁻¹ DW) and flavonoid (6.4 mg g⁻¹ DW) contents were nearly identical to 3-yr-old roots and higher than that of 1-yr-old roots under field cultivation.     

Comparative evaluation of modified bioreactors for enhancement of growth and secondary metabolite biosynthesis usingPanax ginseng hairy roots

Hairy root cultures have demonstrated great promise in terms of their biosynthetic capability toward the production of secondary metabolites, but continue to constitute a major challenge with regard to large-scale cultures. In order to assess the possibility of conducting mass production of biomass, and the extraction of useful metabolites fromPanax ginseng. P. ginseng hairy roots, transformed byRhizobium rhizogenes KCTC 2744, were used in bioreactors of different types and sizes. The most effective mass production of hairy roots was achieved in several differently sized air bubble bioreactors compared to all other bioreactor types. Hairy root growth was enhanced by aeration, and the production increased with increasing aeration rate in a 1 L bioreactor culture. It was determined that the hairy root growth rate could be substantially enhanced by increases in the aeration rate upto 0.5 wm, but at aeration rates above 0.5 wm, only slight promotions in growth rates were observed. In 20 L air bubble bioreactors, with a variety of inoculum sizes, the hairy roots exhibited the most robust growth rates with an inoculum size of 0.1% (w/v), within the range 0.1 to 0.7% (w/v). The specific growth rates of the hairy roots decreased with increases in the inoculum size.     

Mist trickling bioreactor for <Emphasis Type="Italic">Centaurium erythraea</Emphasis> Rafn growth of shoots and production of secoiridoids

Shoots of Centaurium erythraea Rafn were cultivated in 5 l mist trickling bioreactor for 21 and 28 days increasing their dry weight from 0.54 g to 13.7 g and 18.3 g, respectively. About 6880 shoots from 223 initial shoot-tips in 21-day bioreactor producing cycle were produced. The shoots could be successfully rooted and transferred to soil. Secoiridoid accumulation (expressed as a sum of gentiopicroside, sweroside and swertiamarin) in shoots after 21 days of culture reached about 303 mg l⁻¹.     

Movement and containment of microbial contamination in the nutrient mist bioreactor

Summary In vitro plant cultures tend to get contaminated easily with bacteria and fungi because they are grown for long times in sugar-rich media. Contamination of bioreactors is particularly problematic as larger volumes entail larger losses. To study the movement and develop subsequent control of contaminants in the mist bioreactor, the spore-forming microbes Penicillium chrysogenum and Bacillus subtilis were deliberately inoculated into three possible locations in the reactor: the growth chamber (GC), the medium reservoir (R), or the mist-generating chamber (MG). Compared to inoculation into either R or MG regions, the growth of P. chrysogenum inoculated into the GC required 3 more days (c. 60% more time) to move throughout the rest of the reactor. In contrast, regardless of where B. subtilis was inoculated (GC, R, or MG), it took 7d to contaminate the entire system. The movement of filamentous fungi and bacteria seems to follow the same route of contamination throughout this reactor. Once visibly present in the reactor, neither contaminant was controllable by addition of the biocide, Plant Preservative Mixture (PPM). Both microbes were completely inhibited if PPM was added to the MG at the time of inoculation and then again 2-d post-inoculation of plants. Reactors were fun for 3 wk. Plants remained free of contamination. These results will prove useful in the implementation of large-scale in vitro culture systems.     

Plant regeneration via somatic embryogenesis from suspension cell cultures of Lilium×formolongi hort. Using a bioreactor system

Summary In vitro seedlings of Lilium × formolongi Hort. evs. Norikula, RaiZen No. 1, RaiZen No. 3, RaiZen Early, and Bailansa were used to induce callus by variously modified Murashige and Skoog (MS) media, using protocols for flask culture and bioreactor culture. Green embryogenic callus proliferated from roots near the base of bulblets of five varieties on media containing 0.53–5.3 μM α-naphthaleneacetic acid (NAA), and 28 cell lines were obtained by subcultures on the same medium. For flask culture, the fresh weight (FW) of embryogenic cell clumps doubled every 4 wk on MS basal salts supplemented with 0.53°M NAA and 30 g l⁻¹ sucrose. The maximum frequency of somatic embryos that developed into plantlets was 76.67±17% when plated onto solid MS basal medium without plant growth regulators (PGRs). Among the treatments using four types of bioreactors, the best cell growth and regeneration rate (74±0.14%) of somatic embryos was in a modified 2–1 bioreactor. Cells incubated in the other three bioreactors furned brown and died. Histological study revealed that regeneration was by somatic embryogenesis. The regenerants showed normal growth and flowering after 8–9 mo, in the field. A cell line of cv. Norikula has been subcultured in MS basal salts containing 0.53 μM NAA every 2 mo. for 6 yr. The cell aggregates became more synchronous and many typical embryogenic cells with dense cytoplasm were observed under a light microscope. The long-term embryogenic cells plated on MS basal medium still gave rise to numerous somatic embryos and converted into plantlets.     

不同磷、硫及二氧化碳浓度对标志链带藻生长和碳水化合物积累的影响

【目的】为了研究不同磷、硫及二氧化碳浓度对标志链带藻(Desmodesmus insignis)生长与碳水化合物积累的影响,本实验以改良BG11培养基为基础,设计了8种不同初始K_2HPO_4浓度、8种不同初始MgSO_4浓度及4种二氧化碳浓度培养标志链带藻。【方法】采用干重法和苯酚-硫酸法分别测定其生物质浓度与总碳水化合物的含量。【结果】实验结果显示,在高磷浓度(0.460 mmol/L)下生物量达到最高为6.37 g/L,磷浓度为0.230 mmol/L (对照组)时总碳水化合物含量及单位体积产率达到最高,分别为45.40%(%干重)和0.20 g/(L·d)。不同初始MgSO_4浓度实验结果显示,高硫浓度有利于标志链带藻生长及碳水化合物的积累,生物量、总碳水化合物含量及单位体积产率分别在硫浓度为1.217 mmol/L、0.609 mmol/L和1.824 mmol/L时达到最高,分别为7.02 g/L、51.6%(%干重)及0.26 g/(L·d)。当二氧化碳浓度为3%(V/V)时,标志链带藻生物量、总碳水化合物含量及单位体积产率均达到最高,分别为6.81 g/L、44.03%和0.20 g/(L·d)。【结论】因此,磷浓度为0.230 mmol/L、硫浓度为1.824 mmol/L和二氧化碳浓度为3%时最有利于标志链带藻生长及碳水化合物的积累。