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.     

Light-enhanced caffeic acid derivatives biosynthesis in hairy root cultures of <Emphasis Type="Italic">Echinacea purpurea</Emphasis>

Light plays an important role in almost all plant developmental processes and provides the fundamental building blocks for growth, development, primary and secondary metabolism. The effects of light on growth rate and caffeic acid derivative (CADs) biosynthesis in hairy root cultures of Echinacea purpurea (Moench) were assessed. Light-grown hairy roots accumulated increased levels of anthocyanins, which became visible in outer cell layer of the cortex as a ring of purple color. The light-grown root cultures also had radially thickened morphology compared with the dark-grown controls. The growth rate and cell viability of the hairy root cultures in light did not show obvious difference in comparison with those in dark. However, biosynthesis of CADs including cichoric acid, caftaric acid, chlorogenic acid and caffeic acid was significantly increased in hairy root cultures grown in the light. The enhanced accumulation of CADs and anthocyanins in E. purpurea hairy root cultures was correlated to an observed light-stimulated activity of phenylalanine ammonium lyase (PAL).     

Caffeic acid derivatives from a hairy root culture of Lactuca virosa

In a search for biologically active phenolics, a hydroalcoholic extract from the hairy roots of Lactuca virosa was fractionated by chromatographical methods. The procedure led to the isolation of a substantial amount of 3,5-dicaffeoylquinic acid (3,5-DCQA)—a potent free radical scavenger. An analytical RP-HPLC separation of the hydroalcoholic extract from the hairy roots allowed identification of further hydroxycinnamates: caftaric acid (CTA), chlorogenic acid (5-CQA) and cichoric acid (DCTA), as well as small amounts of unbound phenolic acids. A time course of growth and caffeic acid derivatives accumulation in the hairy root culture was also investigated. The highest contents of the compounds in the examined roots were detected at the logarithmic phase of growth. The average content of 3,5-DCQA in the roots (ca. 2.5% DW) was at least one order of magnitude higher than that found in roots of Lactuca species and callus culture of L. virosa.     

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.     

Occurrence of circadian rhythms in hairy root cultures grown under controlled conditions

Hairy roots obtained by transformation via Agrobacterium rhizogenes provide an artificial plant material devoid of aerial parts with high growth on hormone-free media. Fundamental knowledge of hairy root physiology is essential to develop and control its culture. In contrast to shake-flask cultures, a bioreactor set-up combined with on-line data logging provides an efficient tool to study rapid physiological variations in hairy root cultures. Datura innoxia hairy roots were grown in a bioreactor equipped with on-line data analyses of pH, dissolved oxygen (pO2), conductivity, oxygen, and carbon dioxide. The experiments were done at a constant temperature and in the absence of light cues. The results obtained showed that the carbon dioxide evolution rate (CER) presented regular oscillations during the culture. Similar oscillations were also observed for the oxygen uptake rate (OUR). These signals were treated mathematically to look for the existence of a rhythm. An autocorrelation function was used to detect any periodic components. The results demonstrate that hairy root respiration exhibited peaks of 1 day. These oscillations, having a period of about 24 h, were also observed in pH and conductivity signals, although not for the pO2 signal. The data acquired in the absence of hairy roots showed that the observed periodic behavior was not an artifact. No effect on rhythms was observed by the imposition of an external "day/night" cycle. The fact that oscillations persisted in the absence of external stimuli, with a free-running period of 24 h, suggests that a circadian rhythm exists in hairy roots of D. innoxia.     

Root hairiness: effect on fluid flow and oxygen transfer in hairy root cultures

The effect of root hairiness on fluid flow and oxygen transfer in hairy root cultures was investigated using wild-type, transgenic and root-hair mutants of Arabidopsis thaliana. The root hair morphologies of the A. thaliana lines were hairless, short hairs, moderately hairy (wild-type) and excessively hairy, and these morphologies were maintained after transformation of seedlings with Agrobacterium rhizogenes. Filtration experiments were used to determine the permeability of packed beds of roots; permeability declined significantly with increasing root hairiness as well as with increasing biomass density. Hairy roots of wild-type A. thaliana grew fastest with a doubling time of 6.9 days, but the hairless roots exhibited the highest specific oxygen uptake rate. In experiments using a gradientless packed bed reactor with medium recirculation, the liquid velocity required to eliminate external mass transfer boundary layer effects increased with increasing root hairiness, reflecting the greater tendency towards liquid stagnation near the surface of roots covered with hairs. External critical oxygen tensions also increased with increasing root hairiness, ranging from 50% air saturation for hairless roots to ca. 150% air saturation for roots with excessive root hairs. These results are consistent with root hairs providing a significant additional resistance to oxygen transfer to the roots, indicating that very hairy roots are more likely than hairless roots to become oxygen-limited in culture. This investigation demonstrates that root hairiness is an important biological parameter affecting the performance of root cultures and suggests that control over root hair formation, either by use of genetically modified plant lines or manipulation of culture conditions, is desirable in large-scale hairy root systems.     

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.     

Overexpression of alcohol dehydrogenase or pyruvate decarboxylase improves growth of hairy roots at reduced oxygen concentrations.

Overexpression of Arabidopsis thaliana genes for the fermentation enzymes, alcohol dehydrogenase and pyruvate decarboxylase, improved the tolerance of A. thaliana hairy roots to low oxygen conditions. Whereas the specific growth rate of untransformed hairy roots in shake flasks and in a multiple-tube recirculation bioreactor declined significantly with decreasing oxygen tension down to 25% air saturation, growth of the transformant root lines was maintained at rates similar to those achieved with full aeration. This work demonstrates that altering the expression of selected genes involved in anaerobic metabolism can alleviate the problems of oxygen deficiency in hairy root cultures caused by poor mixing and mass transfer conditions.     

Continuous production of scopolamine by a culture of Duboisia leichhardtii hairy root clone in a bioreactor system

The scopolamine-releasing hairy root clone DL47-1 of Duboisia leichhardtii was cultured in an Amberlite XAD-2 column-combined bioreactor system for continuous production of scopolamine. The medium used was continuously exchanged during culture to maintain the electrical conductivity of the medium constant. After culturing the hairy roots in the system for 11 weeks, 0.5 g/l of scopolamine was obtained in the column. When the roots were cultures in the reactor system containing polyurethane foam or stainless-steel mesh to support the hairy roots, scopolamine recovery was increased. Thereafter, a two-stage culture, the first stage in the medium for hairy root growth and the second stage in the medium for scopolamine release, was carried out in this system by using a turbine-blade reactor with stainless-steel mesh as a support. Under these conditions, 1.3 g/l of scopolamine was recovered during 11 weeks of culture in the medium for scopolamine release. This bioreactor system seems applicable for the production of various plant metabolites by cultures of hairy roots. Correspondence to: T. Muranaka     

Inhibitory role of root hairs on transport within root culture bioreactors

An experimental system was developed to produce root cultures of Hyoscyamus muticus with and without the profuse root hairs. Growth in the presence of 7.6 microM pyrene butyric acid (PBA) and 2.2 mM phosphate virtually eliminated root hairs, whereas growth rate, general morphology and nutrient yields remained unchanged in well-mixed flask culture. These root cultures were used to demonstrate decreased flow resistance in a tubular reactor as a result of root hair removal. To assess the impact on bioreactor performance, hairy and hairless root cultures were grown in a highly characterized 15-L bubble column bioreactor. In the absence of root hairs, the mixing was greatly enhanced; mixing times became shorter for the hairless culture at roughly 100 g (fresh weight)/L. By the end of the 3-week culture period, the mixing time of the hairy culture was 29 times longer than that of the hairless culture. The growth rate of the hairless culture in the bioreactor was as much as 2.4 times greater than growth of the hairy culture under the same conditions. The improved reactor performance was reflected in greater biomass accumulation and respiratory activity. These results show that the root hairs-which facilitate nutrient uptake in a static soil environment-are detrimental to growth in a liquid environment as an effect of their stagnating fluid flow and limiting oxygen availability.     

Ri质粒介导的毛状根体系建立及其在植物次生代谢产物合成中的研究进展

发根农杆菌Ri质粒可诱导植物产生毛状根体系,该体系具有遗传性状稳定且增殖速度快的特点,可用于药用植物次生代谢产物的生产研究,为利用生物反应器技术进行药用植物有效成分工业化水平的发酵培养开辟了新途径。本文主要综述了发根农杆菌Ri质粒介导的植物毛状根体系遗传转化机理,并对毛状根体系在药用植物次生代谢产物生产中的研究现状进行了深入分析,为从基因水平上调控植物次生代谢产物的合成提供新思路。     

Transgenic hairy roots. recent trends and applications

Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic roots produced by A. rhizogenes infection is characterized by high growth rate and genetic stability. These genetically transformed root cultures can produce higher levels of secondary metabolites or amounts comparable to that of intact plants. Hairy root cultures offer promise for production of valuable secondary metabolites in many plants. The main constraint for commercial exploitation of hairy root cultures is their scaling up, as there is a need for developing a specially designed bioreactor that permits the growth of interconnected tissues unevenly distributed throughout the vessel. Rheological characteristics of heterogeneous system should also be taken into consideration during mass scale culturing of hairy roots. Development of bioreactor models for hairy root cultures is still a recent phenomenon. It is also necessary to develop computer-aided models for different parameters such as oxygen consumption and excretion of product to the medium. Further, transformed roots are able to regenerate genetically stable plants as transgenics or clones. This property of rapid growth and high plantlet regeneration frequency allows clonal propagation of elite plants. In addition, the altered phenotype of hairy root regenerants (hairy root syndrome) is useful in plant breeding programs with plants of ornamental interest. In vitro transformation and regeneration from hairy roots facilitates application of biotechnology to tree species. The ability to manipulate trees at a cellular and molecular level shows great potential for clonal propagation and genetic improvement. Transgenic root system offers tremendous potential for introducing additional genes along with the Ri T-DNA genes for alteration of metabolic pathways and production of useful metabolites or compounds of interest. This article discusses various applications and perspectives of hairy root cultures and the recent progress achieved with respect to transformation of plants using A. rhizogenes.     

Investigation of liquid-solid hydrodynamic boundary layers and oxygen requirements in hairy root cultures.

Rates of oxygen uptake, growth and alkaloid production by hairy roots in submerged culture were investigated using a recirculation reactor allowing operation at high liquid velocities for removal of hydrodynamic boundary layers. Measurements were performed at dissolved oxygen tensions of 31-450% air saturation. Critical oxygen concentrations for Atropa belladonna hairy roots were above air saturation, viz. 100-125% air saturation for oxygen uptake and 150% air saturation for growth, demonstrating that these roots cultivated in reactors with air sparging are oxygen-limited. The critical oxygen tension for oxygen uptake by Solanum aviculare hairy roots was 75% air saturation. Both the specific oxygen uptake rate and specific growth rate of A. belladonna hairy roots were dependent on the mass (g dry weight) of roots present; even in the absence of boundary layers, growth did not remain exponential over the entire culture period. Cryo-scanning electron microscopy showed that hairy roots grown submerged in liquid medium were covered with thick layers of hydrated mucilage and root hairs, representing a significant additional barrier to oxygen transfer. Roots protruding out of the liquid medium showed no evidence of mucilage accumulation. The specific oxygen demand of A. belladonna root tips was 3.3-11.5 times higher than for the remainder of the roots, the ratio increasing as the dissolved oxygen tension was reduced. Specific growth rates, biomass yields from sugar, and atropine levels were maximum at around 150% air saturation, but decreased significantly with oxygen concentrations above ca. 200%.     

人参毛状根生物合成熊果苷的分离与鉴定

熊果苷(arbutin),化学名称为对-羟基苯-β-D-吡喃葡萄糖苷,能够竞争性抑制酪氨酸酶的活性从而抑制黑色素的形成,被国际公认为高效祛斑美白剂,是化妆品中理想的添加成分.人参(Panax ginseng C. A. Mey.)自古以来就是名贵药材,由于人参在栽培过程中存在着栽培困难、周期过长、地域限制等难题,人参的组织培养受到了广泛的重视.本实验室已建立了人参细胞大量培养体系[1]和人参毛状根培养体系[2],并把熊果苷与人参细胞配伍应用到化妆品生产中,产品深受广大消费者青睐.用植物培养物对外源底物进行生物转化,从而对其结构进行修饰,以获得更有意义的产物的研究报道很多[3～9],也是当今研究的热点.本实验室已对人参生物转化熊果苷的基本条件进行了初步探讨[10],本文在此基础上,对转化产物进行了分离鉴定.     

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。     

Bioreactor production of camptothecin by hairy root cultures of Ophiorrhiza pumila

A large-scale culture of hairy root of Ophiorrhiza pumila using a modified 3 l bioreactor was established. The hairy roots, incited by infection of Agrobacterium rhizogenes were grown in the bioreactor equipped with a stainless net. The final concentration of camptothecin was 0.0085% fresh wt of tissue, and the total production of camptothecin, an anti-neoplastic quinoline alkaloid, reached 22 mg over 8 weeks' culture in the reactor. Approx. 17% (3.6 mg) of the total camptothecin produced was excreted into the culture medium.     

Artemisia tilesii Ledeb hairy roots establishment using Agrobacterium rhizogenes-mediated transformation

An efficient and rapid protocol for the establishment of Artemisia tilesii “hairy” root culture is reported. Leaf explants of aseptically growing plants were cocultured with Agrobacterium rhizogenes A4 wild strain or A. rhizogenes carrying the plasmids with nptII and ifn-α2b genes. Root formation on the explants started in 5–6 days after their cocultivation with bacterial suspension. Prolongation of explant cultivation time on the medium without cefotaxime led to stimulation of root growth. The effects of sucrose concentration as well as of the levels of synthetic indole-3-butyric acid (IBA) and native growth regulator Emistim on the stimulation of A. tilesii “hairy” root growth were studied. Maximum stimulating effect both for the control and for transgenic roots was observed in case of root cultivation on the media supplemented with IBA—up to 7.95- and 9.1-fold biomass increase, respectively. Cultivation on the medium with 10 μl/L Emistime has also led to the control roots growth stimulation (up to 2.75-fold). Emistime at 5 μl/L concentration led to 5.46-fold mass increase in only one “hairy” root line. Higher sucrose content (40 g/L) stimulated growth of two hairy root lines but had no effect on growth of the control roots.     

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.     

Azadirachtin production by hairy root cultivation of Azadirachta indica in a modified stirred tank reactor

Present investigation involves hairy root cultivation of Azadirachta indica in a modified stirred tank reactor under optimized culture conditions for maximum volumetric productivity of azadirachtin. The selected hairy root line (Az-35) was induced via Agrobacterium rhizogenes LBA 920-mediated transformation of A. indica leaf explants (Coimbatore variety, India). Liquid culture of the hairy roots was developed in a modified Murashige and Skoog medium (MM2). To further enhance the productivity of azadirachtin, selected growth regulators (1.0?mg/l IAA and 0.025?mg/l GA₃), permeabilizing agent (0.5?% v/v DNBP), a biotic elicitor (1?% v/v Curvularia (culture filtrate)) and an indirectly linked biosynthetic precursor (50?mg/l cholesterol) were added in the growth medium on 15th day of the hairy root cultivation period in shake flask. Highest azadirachtin production (113?mg/l) was obtained on 25th day of the growth cycle with a biomass of 21?g/l DW. Further, batch cultivation of hairy roots was carried out in a novel liquid-phase bioreactor configuration (modified stirred tank reactor with polyurethane foam as root support) to investigate the possible scale-up of the established A. indica hairy root culture. A biomass production of 15.2?g/l with azadirachtin accumulation in the hairy roots of 6.4?mg/g (97.28?mg/l) could be achieved after 25?days of the batch cultivation period, which was ~27 and ~14?% less biomass and azadirachtin concentration obtained respectively, in shake flasks. An overall volumetric productivity of 3.89?mg/(l?day) of azadirachtin was obtained in the bioreactor.     

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.