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.     

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.     

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.     

Lateral root bridging as a strategy to enhance L-DOPA production in Stizolobium hassjoo hairy root cultures by using a mesh hindrance mist trickling bioreactor

Stizolobium hassjoo hairy roots exhibited a lateral root bridging behavior, enabling not only root dry weight but enhancement of intracellular L-DOPA content. When a single root tip was exerted a proper hindrance, the primary root growth was inhibited while lateral roots were profusely induced. The hindrance-induced lateral roots from individual primary root could bridge together under appropriate inoculation densities, leading to high density hairy root cultures producing secondary metabolites. In the present paper, a novel bioreactor was proposed based on a strategy of lateral root bridging by utilizing mesh as a hindrance, called "mesh hindrance mist trickling bioreactor (MHMTB)". Significant improvements of dry weight and L-DOPA production by using MHMTB were 1.8 and 2.2-folds, respectively, higher than those in the control run without the mesh hindrance within the root bed.     

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.     

Effect of plant growth regulators on growth and biosynthesis of phenolic compounds in genetically transformed hairy roots of<Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer

In this study, morphological alterations, biomass growth, and secondary metabolite production of genetically transformed hairy roots ofPanax ginseng C. A. Meyer, were evaluated after administration of plant growth regulators. The addition of benzylamino purine and kinetin to the culture media increased biomass formation and phenolic compound biosynthesis in the hairy roots. α-Naphthaleneacetic acid and indole-3-butyric acid inhibited hairy root growth, however, low concentrations of indole-3-acetic acid slightly increased hairy root growth. Low concentrations of 2,4-Dichlorophenoxyacetic acid profoundly inhibited growth of hairy roots. The addition of plant growth regulators, such as auxin, did not increase total phenolic compounds in hairy roots that did not contain gibberellic acid and cytokinins. Callus formation was induced in cultures suspended in liquid medium amended with benzylamino purine and kinetin. Hairy roots regenerated from these calluses exhibited an active growth pattern with extensive lateral branching in non-amended medium, similar to the growth pattern of the original hairy roots.     

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.     

Design of an ON/OFF mist duty cycle in mist bioreactors for the growth of hairy roots

In this study, we designed a strategy for selecting the operating conditions of a mist reactor for the growth of hairy roots. This was accomplished by developing a mathematical model to optimize the ON/OFF mist duty cycle for the specified growth of hairy roots. The availability and rate of transport of nutrients to the roots are important parameters in the design and operation of the reactor. The thin liquid film over the root surface, which continuously builds up during the ON cycle, is a major factor that limits mass transfer. The same film also acts as a finite reservoir of nutrients in the absence of any replenishment during the OFF cycle. This reservoir gets depleted as growing roots continue to consume the nutrients. As a result of this depletion the duration of the OFF cycle must be limited such that the nutrient concentration does not go below the critical value required for the specified growth rate. The depleted reservoir is then replenished during the next ON cycle to an extent that depends on feed concentration and duration of the next ON cycle. It was shown that the use of increasing feed concentrations in the fed-batch mode of operation could maintain a specified growth rate in the reactor. Interestingly, this also resulted in the efficient operation of the reactor whereby the reactor operated at slightly above the required concentration and close to the point of minimum mass transfer resistance.     

Computational fluid dynamics modeling of mass transfer behavior in a bioreactor for hairy root culture. I. Model development and experimental validation

A two-dimensional axisymmetric computational fluid dynamics (CFD) model based on a porous media model and a discrete population balance model was established to investigate the hydrodynamics and mass transfer behavior in an airlift bioreactor for hairy root culture.During the hairy root culture of Echinacea purpurea, liquid and gas velocity, gas holdup, mass transfer rate, as well as oxygen concentration distribution in the airlift bioreactor were simulated by this CFD model. Simulative results indicated that liquid flow and turbulence played a dominant role in oxygen mass transfer in the growth domain of the hairy root culture. The dissolved oxygen concentration in the hairy root clump increased from the bottom to the top of the bioreactor cultured with the hairy roots, which was verified by the experimental detection of dissolved oxygen concentration in the hairy root clump. This methodology provided insight understanding on the complex system of hairy root culture and will help to eventually guide the bioreactor design and process intensification of large-scale hairy root culture.     

生物反应器与药用植物毛状根的大规模培养

利用药用植物毛状根培养生产次生代谢产物具有极大的生产潜力,而开发适合毛状根培养的反应器,又是毛状根生产天然产物工业化的关键。本文系统地介绍了各种用于毛状根培养的生物反应器,对通气搅拌式、气升式、超声雾化式等生物反应器各自的特点及优势进行了详细阐述,进一步讨论了各种反应器对于毛状根生长和次生代谢物积累的影响,并提出药用植物毛状根大规模培养的生物反应器在今后的发展方向。     

Influence of hairy root ecotypes on production of tropane alkaloids in Brugmansia candida

Hyoscyamine and scopolamine are tropane alkaloids widely applied in medicine. Differences in alkaloid production and growth kinetics have been observed in Argentinian and Colombian ecotypes of Brugmansia candida hairy roots. The aim of this work was to analyze the production of key intermediates in tropane alkaloid synthesis in both ecotypes to determine differences in the biosynthetic pathway. Additionally, rolC gene expression was analyzed to determine its correlation with hairy root growth. The results showed a higher accumulation of polyamines in Colombian hairy roots, suggesting that there may be a rate-limiting enzyme in the last steps of hyoscyamine biosynthesis. Additionally, rolC gene expression was correlated with an improvement in hairy root growth, which supports the function of rol genes as growth modulators and suggests that metabolic engineering approaches involving rolC manipulation may be useful for the development of more efficient B. candida hairy root cultures for biotechnological applications.     

Transformed root cultures for biotechnology

This review is concerned with the application of hairy roots, i.e. plant roots formed from plant cells after transformation by Agrobacterium rhizogenes for the production of bioactive compounds. Transformed root cultures have been established from numerous species of dicotyledonous plants. The plants, as well as the main products accumulated in hairy root cultures derived from these plants, are listed in this paper. Data are presented on novel compounds, hitherto detected only in transformed roots but not occurring in the corresponding intact plants. The possible use of hairy root cultures for the over-production of secondary metabolites and biotransformation of chemicals is discussed. In order to enhance the productivity of hairy root cultures, various methods have been derived, and optimized procedures are proposed. They include selection of high-producing clones, elicitation, composition of growth media, culture conditions and genetic approach. Hairy roots usually store secondary metabolites in vacuoles inside the cells. Therefore, several methods have been used to increase the amount of products released into the medium. Unfortunately, no general procedure is known that works in all cases, and the excretion behaviour of hairy root cultures varies from one species to another and even within one species from one clone to another. Special attention is given to the cultivation methods and bioreactor systems for hairy root cultures. Hairy roots are cultivated usually in shake flasks; however, shake flask culture is not suitable for the complex optimization and continuous control of the culture conditions. In this paper, we are going to present bioreactors proposed for the cultivation of hairy roots under more or less controlled conditions. Modifications of typical bacterial bioreactors, i.e. stirred tanks, airlift loop reactors and other constructions, are presented. A very special type of bioreactor providing good conditions for loose root mass multiplication without oxygen or substrate limitations, is the mist bioreactor. Nowadays, it is practically impossible to select the one best bioreactor type for hairy root culture.     

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.     

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     

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.     

光对商陆发状根生长和PAP基因表达的影响

利用发根农杆菌菌株Ar1334与美洲商陆(Phytolacca americana)叶片外植体共培养转化体系,共获得58个发状根无性系(SL-1~58).以发根农杆菌Ri质粒TL-DNA上的rol C基因设计特异引物,对发状根进行PCR检测,得到了预期的560 bp目的片段,表明Ri质粒T-DNA整合到发状根基因组中.将筛选出的株系SL-7接种在MS培养基上分别置于光、暗条件下进行培养.结果发现:SL-7在暗培养条件下呈乳白色,具有多分枝、多根毛、无向地性等典型的发状根特性;在光培养条件下,发根呈粉红色,少分支且生长缓慢;以商陆抗病毒蛋白(pokeweed antiviral protein,PAP)cDNA片段为探针,分别对光、暗条件下的发状根进行Northern blot检测,发现光对PAP基因的转录具有一定抑制作用;将发状根粗蛋白提取液与TMV病毒液混合后,摩擦接种于心叶烟(Nicotiana glutinosa)离体叶片,发现暗培养的发状根粗蛋白提取液对TMV抗性明显提高.表明商陆发状根的生长及PAP基因的表达都受到光的负向调控.该结果为商陆发状根的规模化培养和PAP蛋白的离体合成优化体系的建立奠定了基础.     

A flow cell reactor for the study of growth kinetics of single hairy roots

Summary A flow cell reactor designed for the study of growth and branching of individual roots under defined fluid flow conditions is described and preliminary results with the hairy root clone Tagetes erecta T3 are presented. Observations of root growth in this reactor may help to formulate and verify mathematical models root growth kinetics.     

金铁锁毛状根构型对其生长的影响

毛状根的构型是影响其生长速度和生物量积累的重要因素,为了规模化培养金铁锁毛状根,进一步解决金铁锁资源短缺问题,该研究以金铁锁毛状根为材料,通过改变培养基类型、碳源及碳源浓度,观察和分析了毛状根的生长状态,找出影响毛状根构型的因素。结果表明:最适合金铁锁毛状根生长的培养基为B5+蔗糖30 g·L~(-1),金铁锁毛状根主根长而粗壮,一级、二级侧根生长量大,根系表面积较大,生长效果最佳。经液体悬浮培养验证,测定毛状根的生长量,与在固体培养基培养的毛状根生长状态基本一致。通过该项研究,优化了培养基中营养成分的配比,实现了金铁锁毛状根的快速生长和生物量的积累。