The biological response of hairy roots to O2 levels in bioreactors

Summary The efficient exchange of gases between roots and their environment is one of the biggest challenges in bioreactor design for transformed root cultures. Gas-phase reactors can alleviate this problem as well as provide a new tool for studying the biological response of roots and other differentiated tissues to changes in the gas phase composition. In our comparison of liquid- and gas-phase reactors, roots grown in liquid (shake flasks or bubble column reactors) are shown to be under hypoxic stress. Roots grown in a gas-phase reactor (nutrient mist), while not hypoxic, produced 50% less biomass. These results suggest that the response of the tissues to gas phase composition are complex and need further study.     

Growth of Artemisia annua hairy roots in liquid- and gas-phase reactors

Artemisia annua hairy roots were grown in liquid-phase bubble column and gas-phase nutrient mist reactors. In most cases the bubble column reactor accumulated more biomass than the mist reactor; the highest final biomass concentrations observed were 15.3 g DW/L in the bubble column reactor and 14.4 g DW/L in the mist reactor. Further analysis showed that the average specific growth rate in the mist reactors was essentially constant and independent of the biomass concentration at the beginning of the mist mode. In contrast, at low packing densities the average growth rate in the bubble column reactors was higher than in the mist reactors, decreasing to comparable rates at high packing densities. Finally, an aerosol deposition model was used to compare the volume of medium captured by the root bed in the mist reactor to the volume of medium required to maintain a specified growth rate. The results suggest that under the current operating conditions, lower growth rates in the mist reactor may be due to insufficient nutrient availability.     

Biomass production of hairy roots of Artemisia annua and Arachis hypogaea in a scaled‐up mist bioreactor

Hairy roots have the potential to produce a variety of valuable small and large molecules. The mist reactor is a gas phase bioreactor that has shown promise for low‐cost culture of hairy roots. Using a newer, disposable culture bag, mist reactor performance was studied with two species, Artemisia annua L. and Arachis hypogaea (peanut), at scales from 1 to 20 L. Both species of hairy roots when grown at 1 L in the mist reactor showed growth rates that surpassed that in shake flasks. From the information gleaned at 1 L, Arachis was scaled further to 4 and then 20 L. Misting duty cycle, culture medium flow rate, and timing of when flow rate was increased were varied. In a mist reactor increasing the misting cycle or increasing the medium flow rate are the two alternatives for increased delivery of liquid nutrients to the root bed. Longer misting cycles beyond 2–3 min were generally deemed detrimental to growth. On the other hand, increasing the medium flow rate to the sonic nozzle especially during the exponential phase of root growth (weeks 2–3) was the most important factor for increasing growth rates and biomass yields in the 20 L reactors. A. hypogaea growth in 1 L reactors was µ = 0.173 day^?1 with biomass yield of 12.75 g DW L^?1. This exceeded that in shake flasks at µ = 0.166 day^?1 and 11.10 g DW L^?1. Best growth rate and biomass yield at 20 L was µ = 0.147 and 7.77 g DW L^?1, which was mainly achieved when medium flow rate delivery was increased. The mist deposition model was further evaluated using this newer reactor design and when the apparent thickness of roots (+hairs) was taken into account, the empirical data correlated with model predictions. Together these results establish the most important conditions to explore for future optimization of the mist bioreactor for culture of hairy roots. Biotechnol. Bioeng. 2010;107: 802–813. © 2010 Wiley Periodicals, Inc.     

Growth and Pigment Production by Hairy Root Cultures of Beta Vulgaris L. in a Bubble Column Reactor

Beet hairy root cultures established from red and yellow varieties were grown in a 2 L bubble column reactor. The yellow clone showed profuse root hairs and a predominance of betaxanthin pigment with the red clone showed fewer root hairs and both betaxanthin and betacyanin pigments. The cultures displayed different ionic and sugar yields: 2.1 mg dry wt / mS.mL and 0.361 g dry wt / g sugar for the yellow clone and 2.3 mg dry wt / mS.mL and 0.375 g dry wt / g sugar for the red one. Both cultures grew at the same specific growth rate of 0.22 d^-1in the bubble column, as compared to 0.32 d^-1in shake flasks, indicating mass transfer limitations for growth in reactors.     

A comparative study of mist and bubble column reactors in the in vitro production of artemisinin

Artemisia annua hairy roots grown in nutrient mist reactors produced nearly three times as much artemisinin as roots grown in bubble column reactors, 2.64 µg/g DW and 0.98 µg/g DW, respectively.     

Transformed roots of Artemisia annua exhibit an unusual pattern of border cell release

Summary Border cells from Artemisia annua were examined from hairy roots grown in shake flasks, culture plates, a bubble column reactor, and a nutrient mist (aeroponic) reactor. When well-hydrated roots were subjected to shear, border cells were first released as an agglomerate and did not disperse for several hours. Staining with neutral red and fluorescein diacetate (FDA) showed that both agglomerates and dispersed cells were alive. It was determined that FDA is cleaved by pectin methylesterase (PME) and that PME may not be particularly active in the released agglomerates until the border cells disperse. Untransformed roots isolated from A. annua plants showed no border cell agglomerate formation and border cells readily dispersed. These results suggest that our hairy root clone is deficient in border cell release perhaps resulting from the transformation process.     

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.     

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.     

Process-engineering characterization of small-scale bubble columns for microbial process development

Volumetric oxygen transfer rates and power inputs were estimated by a model of the formation of primary gas bubbles at the static sparger (sinter plate) of small-scale bubble columns and a common mass-transfer correlation for bubbles rising in a non-coalescent Newtonian electrolyte solution of low viscosity. Estimations were used to assess the dimensioning and possibilities of small-scale bubble column application with an height/diameter ratio of about 1. Estimations of volumetric oxygen transfer rates (<0.16 s^-1) and power inputs (<100 W m^-3) with a mean pore diameter of the static sparger of 13 µm were confirmed as function of the superficial air velocity (<0.6 cm s^-1) by measurements using an Escherichia coli fermentation medium. Small-scale bubble columns are thus to be classified between shaking flasks and stirred-tank reactors with respect to the oxygen transfer rate, but the maximum volumetric power input is more than one magnitude below the power input in shaking flasks, which is of the same order of magnitude as in stirred-tank reactors. A small-scale bubble columns system was developed for microbial process development, which is characterized by handling in analogy to shaking flasks, high oxygen transfer rates and simultaneous operation of up to 16 small-scale reactors with individual gas supply in an incubation chamber.     

Effect of bioreactor configuration on the growth and maturation of Picea sitchensis somatic embryo cultures

Somatic embryo suspension cultures of Picea sitchensis (Sitka spruce) derived from two cell lines, SS03 and SS10, were grown in shake flasks, air-lift, bubble, stirred tank and hanging stirrer bar bioreactors. Cell line SS03 yielded freely suspended and individual stage 1 embryos, while the embryos of SS10 were present in large aggregates. Compared to shake flasks, proliferation in bioreactors resulted in increased biomass; however, cell line morphology influenced the effect of different bioreactor configurations on growth and maturation of embryo cultures. Somatic embryos grown in shake flasks and bioreactors were matured on gelled solid medium and in submerged culture where gelled solid medium was covered with a layer of liquid medium. The number of stage 3 (mature) embryos produced from SS03 in the bubble bioreactor was significantly higher than those from stirred tank and hanging stirrer bar bioreactors with both solid medium and submerged culture. Submerged culture was unsuitable for SS10 embryo maturation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Indole alkaloid production by hairy root cultures ofCatharanthus roseus: Growth kinetics and fermentation

Summary Growth kinetics and indole alkaloid production ofCatharanthus roseus hairy root cultures were studied in shake flasks and in a small scale fermenter. A logistic growth model commonly used for microbes described well the growth of hairy roots. Of the several parameters analyzed during the cultivation of hairy roots, a linear relationship between sucrose consumption and dry weight increase was obtained. This suggests the validity of sugar analysis as a means in monitoring the growth of hairy roots in fermenters.     

Using an aerosol deposition model to increase hairy root growth in a mist reactor

Gas-phase reactors, including the mist reactor, have distinct advantages over liquid-phase reactors including the ability to manipulate the gas composition, to allow effective gas exchange in a densely growing biomass, and to affect secondary metabolite production. Mathematical modeling suggested that roots in a mist reactor are often too sparsely packed to capture mist particles efficiently and cannot, therefore, meet the nutrient demands required to maintain high growth rates. Indeed, growth rates of Artemisia annua hairy roots increased significantly when the initial packing density increased or when a higher sucrose concentration was used in the medium. Growth kinetics for 2, 4, and 6 days, however, showed a decrease or stationary growth rate after only 4 days for both 3 and 5% sucrose feeds. Residual medium analyses indicated that carbon was not exhausted, nor were any of the other major nutrients including phosphate. Increasing the mist duty cycle at constant carbon flux through the reactor reduced the growth rates slightly. In general, the aerosol deposition model correctly predicted how to optimize hairy root growth in the mist reactor.     

Monitoring biomass in root culture systems

This paper presents a technique for accurate estimation of growth in root culture systems. Biomass correlations, were used to estimate fresh weight time course data in shake flasks and reactors based on a model of liquid nutrient uptake and osmolality, to account for changing specific water content of roots. This mass balance technique has been developed to permit accurate aseptic on-line estimation of dry weight (DW), fresh weight (FW), and liquid volume (V) in root cultures utilizing either refractive index or electrical conductivity of the liquid medium along with liquid medium osmolality. The ability to predict fresh weight is particularly important since this is proportional to the biomass volume fraction which determines mass transfer and other culture transport characteristics. The proposed model has been validated with time course information (DW, FW, and V) from 125 mL shake flasks and corroborated with data obtained from 2 L reactors. Copyright 1999 John Wiley & Sons, Inc.     

Effects of the oxygen transfer rate on ferrous iron oxidation by Thiobacillus ferrooxidans

Ferrous iron oxidation by Thiobacillus ferrooxidans was studied in shake flasks and a bubble column under different aeration conditions. The maximum biooxidation rate constant was affected by oxygen transfer only at low aeration intensities. At oxygen transfer rates higher than 0.03 mmol O₂ l⁻¹ min⁻¹, the maximum biooxidation rate constant was about 0.050 h⁻¹ in both shake flasks of different size and the bubble column. The oxygen transfer rate could be used as a basis for scaling up bioreactors for ferrous iron biooxidation by T. ferrooxidans.     

Comparison of the growth ofCinchona ledgeriana Moens suspension cultures in shake flasks and 7 litre air-lift bioreactors

Summary Suspension cultures ofCinchona ledgeriana Moens have been developed which exhibit good growth in shake flasks with dry weight yields of approximately 9.0 g.l^–1. Cultures have been scaled up for growth in a 7 l air-lift bioreactor. A typical growth curve in the fermenter is shown with similar growth rates but a reduced biomass levels when compared to shake flasks. The analysis of both flask and bioreactor grown suspension cultures indicated the presence of quinidine and low levels of quinine.     

Novel airlift reactor fitting for hairy root cultures: developmental and performance studies

In vitro growth of Solanum chrysotrichum hairy roots was carried out in three different types of reactors: shake flasks, a glass-draught internal-loop 2-L basic design airlift reactor (BDR), and a novel modified mesh-draught with wire-helixes 2-L reactor (MR). In each of them, the growth patterns were different, as well as some of the dynamic parameters. The specific growth rates were 0.08, 0.067, and 0.112 d(-1) for shake flasks, BDR, and MR, respectively. In shake flasks and in the MR, growth followed first-order kinetics. In the MR without roots, superficial liquid velocity in the riser and downcomer ranged from 2.1 to 2.7 and 1.4 to 1.7 cm s(-1), respectively (nearly the same as the BDR values). After 42 days in culture, tissue density in the MR was twice that found in the BDR and about the same as that found in the shake flasks. At the tissue densities reached at 42 days, superficial liquid velocities in the MR and BDR downcomers were 4-5 and 7-8 times lower, and mixing times were 11 and 18 times longer than those observed without roots. Tissue densities measured at three points in the MR's downcomer and riser ranged from 10.21 to 12.17 and 4.94 to 5.24 gDW L(-1) respectively. Dynamic gas hold-up dropped faster when roots grew radially in the mesh-draught. In addition, root cultures were scaled-up in a 10-L MR reactor in which some geometric relations were maintained, such as the Q/V radio. Growth in 10-L MR followed first-order kinetics, but despite this, specific growth velocity was 0.09 d(-1) and overall tissue density diminished slightly with respect to that of the 2-L MR. Tissue inoculation, distribution, and harvest were more easily accomplished in the MRs.     

Different bioreactor configurations for the decolourisation of the azo dye reactive black 5 by Geotrichum sp. CCMI 1019

Decolourisation of the azo dye Reactive Black 5 by Geotrichum sp. CCMI 1019 was studied using stirred tank reactors (STR) and two types of bubble columns (porous plate (PP) bubble column and aeration tube (AT) bubble column). For the bubble columns, the k_La increased with the gas fractional hold-up (ε_G) and the aeration rate. A linear relationship between ε_G and superficial gas velocity was obtained for all reactors. At same aeration rates, the PP bubble columns showed higher k_La and hold-up values than the AT bubble column. In the STRs, large and dense aggregates were formed which adhered to surfaces whereas bubble columns gave smaller and less compact pellets.

Manganese peroxidase and laccase were detected in the extracellular media in all reactors. However, laccase was only detected after the onset of decolourisation, suggesting that additional enzymes may be involved. Mn peroxidase activity was detected (about 46 U/ml) in both the STRs and AT bubble columns but higher values (110 U/ml) were obtained with the PP bubble columns.

Out of the three reactor systems studied, the AT bubble columns gave the most favourable results for Reactive Black 5 decolourisation. Rapid and complete colour removal was obtained throughout the visible spectrum. Bubble columns are simple in design as well as operation and may be useful for the bioremediation of textile wastewater.