Selection on tropane alkaloids in native and non‐native populations of Datura stramonium

Theories of plant invasion based on enemy release in a new range assume that selection exerted by specialist herbivores on defence traits should be reduced, absent, or even selected against in the new environment. Here, we measured phenotypic selection on atropine and scopolamine concentration of Datura stramonium in eight native (Mexico) and 14 non‐native (Spain) populations. Native populations produced between 20 and 40 times more alkaloid than non‐native populations (atropine: 2.0171 vs. 0.0458 mg/g; scopolamine: 1.004 vs. 0.0488 mg/g, respectively). Selection on alkaloids was negative for atropine and positive for scopolamine concentration in both ranges. However, the effect sizes of selection gradients were only significant in the native range. Our results support the assumption that the reduction of plant defence in the absence of the plant's natural enemies in invasive ranges is driven by natural selection.     

Hydrodynamics, mass transfer and rheological studies of gibberellic acid production in an airlift bioreactor

Although a lot of research has been done into modelling microbial processes, the applicability of these concepts to problems specific for bioreactor design and optimization of process conditions is limited. This is partly due to the tendency to separate the two essential factors of bioreactor modelling, i.e. physical transport processes and microbial kinetics. The deficiencies of these models become especially evident in industrial production processes where O₂ supply is likely to become the limiting factor, e.g. production of gibberellic acid and other organic acids. Hydrodynamics, mass transfer and rheology of gibberellic acid production by Gibberella fujikuroi in an airlift bioreactor is presented in this work. Important hydrodynamic parameters such as gas holdup, liquid velocity in the riser and in the downcomer, and mixing time were determined and correlated with superficial gas velocity in the riser. Mass transfer was studied evaluating the volumetric mass transfer coefficient, which was determined as a function of superficial gas velocity in the riser and as a function of fermentation time. Culture medium rheology was studied through fermentation time and allowed to explain the volumetric mass transfer coefficient behaviour. Rheological behaviour was explained in terms of changes in the morphology of the fungus. Finally, rheological studies let us obtain correlations for gas holdup and volumetric mass transfer coefficient estimation using the superficial gas velocity in the riser and the culture medium apparent viscosity.     

Cultivation of Arabidopsis cell cultures in a stirred bioreactor at variable oxygen levels: Influence on tocopherol production

Abstract

In plants, an increased production of toxic oxygen species is commonly observed under low oxygen stress, but cellular responses still have to be fully investigated. Plant cell cultures can be a valuable tool to study plant metabolic responses to various environmental stresses including low oxygen condition. Arabidopsis suspension cultures growing in shake flasks were subjected to hypoxia by stopping shaking for different intervals, showing an increase of the antioxidant metabolite α‐tocopherol. In order to obtain a more controlled condition, cultivation of Arabidopsis suspension cultures was established in a 5‐l stirred bioreactor. A constant aeration of 20% dissolved oxygen was found to be the most suitable for cell growth. A 4‐h anoxic shock was induced by suspending the aeration and flushing into the vessel with nitrogen. During the anoxic stress, tocopherol levels resulted increased at the end of the treatment, indicating that the complete oxygen deprivation, indeed, induced a defence response involving antioxidant metabolism. The presence of an oxidative stress as a consequence of anoxic condition was also confirmed by the increased levels of H₂O₂. Overall, these results indicate that Arabidopsis suspension cultures grown in a stirred bioreactor can be a useful in vitro system for investigating low oxygen stress.     

Advantages and limitations in the use of hairy root cultures for the production of tropane alkaloids: Use of anti-auxins in the maintenance of normal root morphology

Summary Brugmansia candida hairy roots, obtained by infection withAgrobacterium rhizogenes LBA 9402, exhibit, after subculturing in liquid media, a tendency towards dedifferentiation. It has been found that the following strategies can be applied to inhibit this dedifferentiation and preserve normal root morphology: (a) lowering both the mineral and sucrose concentration in the media employed so as to diminish osmotic stress (a condition to which these roots appear to be particularly susceptible); (b) employing antiauxins in appropriate concentrations; and (c) maintaining the hairy roots on solid media prior to use in production processes in liquid media. The first strategy suggested does not favor alkaloid productivity, but in this case a two-step method could be attempted: biomass with normal root morphology could be obtained in a first stage using low sucrose concentrations, and in a second stage, sucrose could be increased in order to achieve higher productivity. In all the clones ofB. candida obtained, alkaloid production was biased towards scopolamine.     

Fluid mixing and oxygen transfer in cell suspensions ofTaxus chinensis in a novel stirred bioreactor

In high-density plant cell cultures, mixing and mass transfer are two key issues, which should be emphasized for process optimization. In this work, both mixing and oxygen transfer characteristics of cell suspensions ofTaxus chinensis were studied in a new centrifugal impeller bioreactor with a working volume of 1.2 L. The mixing time (t _M) and the volumetric oxygen transfer coefficient (K _L a) under different operational conditions were determined in both tap water and cell suspensions of 100–400 g fresh weight/L (i.e., 5.65–23.1 g DW/L). At an aeration rate of 0.1 L/min,t _M decreased from 10.6s at 30 rpm to 2.89 s at 200 rpm under 100 g FW/L, and from 9.63 s (120 rpm) to 4.05 s (300 rpm) under 400 g FW/L. Compared with the effect of agitation, aeration was less significant to the suspension mixing. At a relatively high agitation speed (e.g., 200 rpm),t _M remained almost the same even though aeration rate was changed from 0.1 to 0.4 L/min. Thet _M value increased slowly from 3.98 to 5.26 s at 120 rpm when the cell density was raised from 100 to 250 g FW/L. A rapid increase of botht _M and the suspension viscosity was observed at a cell density above 300 g FW/L. As expected, theK _L a value increased with an increase of aeration rate and agitation speed, but decreased with an increase of cell density. The quantitative data obtained here are useful to investigate the effect of mixing stress on the cell physiology and metabolism ofTaxus chinensis in the bioreactor. This paper is dedicated by JJZ to his colleague Prof. Jun-Tang Yu on the occasion of his 70 birthday.     

Characterization and improvement of oxygen transfer in pilot plant external air-lift bioreactor for mycelial biomass production

The oxygen transfer dynamics in a pilot plant external air-lift bioreactor (EALB) during the cultivation of mycelial biomass were characterized with respect to hydrodynamic parameters of gas holdup (), oxygen transfer coefficient (K_La) and superficial gas velocity (U _g), and dissolved oxygen (DO). An increased flow rate of air supply was required to meet the increased oxygen demand with mycelial biomass growth. Consequently, an increase in air flow rate led to an increase in , K_La and the DO level. The enhancement of oxygen transfer rate in the cultivated broth system, however, was limited with highly increased viscosity of the mycelial broth. An increase in air flow rate from 1.25 to 2.00 v/v/m resulted in a low increment of oxygen transfer. The newly designed pilot plant EALB with two air spargers significantly improved processing reliability, aeration rate and K_La. The pilot plant EALB process, operated under a top pressure from 0 to 1.0 bars, also demonstrated a significant improvement of oxygenation efficiency by more than 20% in DO and K_La. The performance of the two sparger EALB process under top pressure demonstrated an efficient and economical aerobic system with fast mycelial growth and high biomass productivity in mycelial biomass production and wastewater treatment.     

Effects of oxygen volumetric mass transfer coefficient and pH on lipase production by Staphylococcus warneri EX17

The principal objectives of this study were to evaluate the kinetics of lipase production by Staphylococcus warneri EX17 under different oxygen volumetric mass transfer coefficients (k_La) and pH conditions in submerged bioreactors, using glycerol (a biodiesel by-product) as a carbon source. Cultivations were conducted at different k_La (26, 38, 50, and 83 h⁻¹) and pH values (6.0, 7.0, and 8.0). The optimal k_La and pH were 38 h⁻¹ and 7.0, respectively. Under these conditions, the maximal cell production obtained was 8.0 g/L, and the volumetric and specific lipase production reached high levels of activity, approximately 800 U/L and 150 U/g cell, respectively, after 12 h of cultivation. This result was approximately five times higher than that obtained in the shake flask cultures. The relationship between cell growth and lipase production was found to be associated with growth by the Luedeking-Piret model.     

Outdoor helical tubular photobioreactors for microalgal production: modeling of fluid-dynamics and mass transfer and assessment of biomass productivity

The production of the microalga Phaeodactylum tricornutum in an outdoor helical reactor was analyzed. First, fluid dynamics, mass-transfer capability, and mixing of the reactor was evaluated at different superficial gas velocities. Performance of the reactor was controlled by power input per culture volume. A maximum liquid velocity of 0.32 m s(-1) and mass transfer coefficient of 0.006 s(-1) were measured at 3200 W m(-3). A model of the influence of superficial gas velocity on the following reactor parameters was proposed: gas hold-up, induced liquid velocity, and mass transfer coefficient, with the accuracy of the model being demonstrated. Second, the influence of superficial gas velocity on the yield of the culture was evaluated in discontinuous and continuous cultures. Mean daily values of culture parameters, including dissolved oxygen, biomass concentration, chlorophyll fluorescence (F(v)/F(m) ratio), growth rate, biomass productivity, and photosynthetic efficiency, were determined. Different growth curves were measured when the superficial gas velocity was modified-the higher the superficial gas velocity, the higher the yield of the system. In continuous mode, biomass productivity increased by 35%, from 1.02 to 1.38 g L(-1) d(-1), when the superficial gas velocity increased from 0.27 to 0.41 m s(-1). Maximal growth rates of 0.068 h(-1), biomass productivities up to 1.4 g L(-1) d(-1), and photosynthetic efficiency of up to 15% were obtained at the higher superficial gas velocity of 0.41 m s(-1). The fluorescence parameter, F(v)/F(m), which reflects the maximal efficiency of PSII photochemistry, showed that the cultures were stressed at average irradiances within the culture higher than 280 microE m(-2) s(-1) at every superficial gas velocity. For nonstressed cultures, the yield of the system was a function of average irradiance inside the culture, with the superficial gas velocity determining this relationship. When superficial gas velocity was increased, higher growth rates, biomass productivities, and photosynthetic efficiencies were obtained for similar average irradiance values. The higher the superficial gas velocity, the higher the liquid velocity, with this increase enhancing the movement of the cells inside the culture. In this way the efficiency of the cells increased and higher biomass concentrations and productivities were reached for the same solar irradiance.     

Influence of sparger on energy dissipation, shear rate, and mass transfer to sea water in a concentric-tube airlift bioreactor

Data on volumetric mass-transfer coefficient, K_La_L, in a 12 × 10⁻³ m³ airlift bioreactor are reported. Measurements were made in sea water. The superficial gas velocity ranged up to 0.21 m/s. Four cylindrical spargers (60–1000 μm pore size) were tested. In bubbly flow, the sparger pore size strongly influenced the K_La_L; the highest K_La_L values were obtained with the smallest pore size. In contrast, in the transition and heterogeneous flow regimes, the pore size had little influence on K_La_L. The best correlation of the mass transfer data was obtained when both gas holdup and liquid superficial velocity were taken as independent variables. Shear rates were estimated in the different zones of the reactor. The highest values were found in the bottom zone of the reactor and in the gas-liquid separator. The penetration and isotropic turbulence models were used to develop a semi-theoretical equation relating the volumetric mass-transfer coefficient to shear rate; hence providing a better understanding of how the operational variables may be manipulated to attain a moderate shear rate and an appropriate level of mass transfer, two extremely important parameters for the growth of sensible microorganisms as those used in marine biotechnology.     

High density cell culture of Panax notoginseng for production of ginseng saponin and polysaccharide in an airlift bioreactor

High cell density of Panax notoginseng in a 17 l airlift bioreactor was achieved in batch cultivation using a modified MS medium. The dry cell weight, ginseng saponin and polysaccharide reached 24, 1.7 and 2.8 g l^–1, respectively, after 15 d. A strategy of sucrose feeding based on changes in the specific O₂ uptake rate was applied to the cell cultures, which increased these respective yields to 30, 2.3 and 3.2 g l^–1.     

Semicontinuous bioreactor production of a recombinant human therapeutic protein using a chemically inducible viral amplicon expression system in transgenic plant cell suspension cultures

Plant cell culture is an alternative for the production of recombinant human therapeutic proteins because of improved product safety, lower production cost, and capability for eukaryotic post‐translational modification. In this study, bioreactor production of recombinant human alpha‐1‐antitrypsin (rAAT) glycoprotein using a chemically inducible Cucumber mosaic virus (CMV) viral amplicon expression system in transgenic Nicotiana benthamiana cell culture is presented. Optimization of a chemically inducible plant cell culture requires evaluation of effects of timing of induction (TOI) and concentration of inducer (COI) on protein productivity and protein quality (biological functionality). To determine the optimal TOI, the oxygen uptake rate (OUR) of the plant cell culture was chosen as a physiological indicator for inducing maximum rAAT expression. Effects of COI on rAAT production were investigated using a semicontinuous culture, which enables the distinction between effects of growth rate and effects of inducer concentration. An optimized semicontinuous bioreactor operation was further proposed to maximize the recombinant protein production. The results demonstrated that the transgenic plant cells, transformed with the inducible viral amplicon expression system, maintain higher OUR and exhibit lower extracellular protease activity and lower total phenolics concentration in the optimized semicontinuous bioreactor process than in a traditional batch bioreactor operation, resulting in a 25‐fold increase in extracellular functional rAAT (603 µg/L) and a higher ratio of functional rAAT to total rAAT (85–90%). Surprisingly, sustained rAAT production and steady state, long‐term bioreactor operation is possible following chemical induction and establishment of the viral amplicons. Biotechnol. Bioeng. 2010; 106: 408–421. © 2010 Wiley Periodicals, Inc.     

Effects of alginate and immobilization by entrapment in alginate on benzophenanthridine alkaloid production in cell suspension cultures of Eschscholtzia californica

The production of benzophenanthridine alkaloids (sanguinarine, chelerythrine and macarpine) in cells of Eschscholtzia californica is enhanced by sodium alginate and by entrapment in Ca2+-alginate. Tyrosine decarboxylase, a key enzyme of alkaloid biosynthesis, is induced by the treatments. Alginate- entrapped cells are elicited over an extended period of time which leads to increased alkaloid biosynthesis (up to 800-fold enhancement). A major portion of alkaloids produced are released into the growth medium.     

水分和盐分胁迫对番茄植株生长和木质部水力特性的影响

木质部是植株体内水分传输的主要通路,其水力特性的变化会影响植株的水分关系和果实的水分积累。目前关于番茄植株木质部解剖结构和水力特性对水分和盐分胁迫的响应及其与植株生长和果实含水量之间的关系尚不明确。本研究通过日光温室番茄盆栽试验,设置3个处理:对照,土壤含水量(θ)为75%～95%田间持水量(FC),初始电导率(EC)为0.398 dS·m^-1;水分胁迫,开花前θ为75%～95% FC,开花后至成熟期θ为45%～65% FC,EC为0.398 dS·m^-1;盐分胁迫,θ为75%～95% FC,EC为1.680 dS·m^-1,研究了樱桃型番茄(红宝石)和中果型番茄(北番501)植株在水分和盐分胁迫下的植株生长、果实含水量以及木质部水力特性的变化。结果表明: 与对照相比,水分和盐分胁迫下茎秆横截面积和木质部导管直径分别减小了22.0%～40.7%和10.0%～18.3%,茎秆比导水率和桁架柄比导水率分别降低了8.8%～41.1%和12.9%～28.4%,抑制了植株生长,减少了地上部鲜重、果实大小、果实鲜重和含水量,且与樱桃型番茄相比,中果型番茄的降幅更大。此外,果实含水量分别与茎秆和桁架柄比导水率呈显著正相关。综上,番茄植株在水分和盐分胁迫下木质部水力特性指标减小,生长被抑制,果实鲜重显著降低,最终导致产量降低。其中,中果型番茄相较于樱桃型番茄对水分和盐分胁迫更敏感。     

Convective-diffusive mass transfer of agonist and the intracellular calcium response of endothelial cell

Endothelial cell layer regulates several crucial physiological processes of the vascular system. The mechanism of the response of this cell layer to the flow of surrounding fluid is still largely unclear. In the present article, a comparison of the available experimental results for the intracellular calcium ion concentration and theoretical results for the extra-cellular ATP concentration obtained using a convective-diffusive mass transfer model, has been made which supports the mass transfer model for the endothelial cell response to the fluid flow. The experimental results are in excellent agreement with the calculated values assuming the effect of ATP concentration alone.     

Understanding the effect of high gas entrance velocity on Chinese hamster ovary (CHO) cell culture performance and its implications on bioreactor scale-up and sparger design

There are three main potential sources for cell shear damage existing in stirred tank bioreactors. One is the potential high energy dissipation in the immediate impeller zones; another from small gas bubble burst; and third is from high gas entrance velocity (GEV) emitting from the sparger. While the first two have been thoroughly addressed for the scale-up of Chinese hamster ovary (CHO) cell culture knowing that a wide tolerable agitation range with non-damaging energy dissipation exists and the use of shear protectants like Pluronic F68 guard against cell damage caused by bubble burst, GEV remains a potential scale-up problem across scales for the drilled hole or open pipe sparger designs. GEV as high as 170 m/s due to high gas flow rates and relatively small sparger hole diameters was observed to be significantly detrimental to cell culture performance in a 12,000 L bioreactor when compared to a satellite 2 L bioreactor run with GEV of <1 m/s. Small scale study of GEV as high as 265 m/s confirmed this. Based on the results of this study, a critical GEV of >60 m/s for CHO cells is proposed, whereas previously 30 m/s has been reported for NS0 cells by Zhu, Cuenca, Zhou, and Varma (2008. Biotechnol. Bioeng., 101, 751–760). Implementation of new large scale spargers with larger diameter and more holes lowered GEV and helped improve the cell culture performance, closing the scale-up gap. Design of such new spargers was even more critical when hole plugging was discovered during large scale cultivation hence exacerbating the GEV impact. Furthermore, development of a scale down model based on mimicry of the large scale GEV profile as a function of time was proven to be beneficial for reproducing large scale results.     

红砂灌丛对土壤和草本植物特征的影响

研究了黄土高原西部荒漠草原区天然红砂灌丛内外土壤水分、养分的差异及植物萌动期红砂灌丛对草本植物生长和植被组成的影响.结果表明:红砂灌丛下土壤水分状况明显好于灌丛外空地,尤其是在30～110 cm土层最为显著;灌丛内土壤粘粒和粉粒含量明显高于灌丛外,而沙粒含量低于灌丛外,尤其是0～10 cm土层;土壤有机质、全效氮、磷、钾及速效氮、磷、钾均在灌丛下富集,富集率分别达到1.40、1.25、1.04、1.05、1.37、1.77和1.49;从灌丛内到灌丛外草本植物的盖度和高度逐渐减小,而植物丰富度增大;红砂灌丛对草本植物密度、盖度和高度的相互作用强度均呈现正值,而植物丰富度的相互作用强度为负值.说明红砂灌丛具有明显的沃岛效应,并对草本植物的生长有促进作用.     

Production of oncolytic adenovirus and human mesenchymal stem cells in a single‐use,Vertical‐Wheel bioreactor system: Impact of bioreactor design on performance of microcarrier‐based cell culture processes

Anchorage‐dependent cell cultures are used for the production of viruses, viral vectors, and vaccines, as well as for various cell therapies and tissue engineering applications. Most of these applications currently rely on planar technologies for the generation of biological products. However, as new cell therapy product candidates move from clinical trials towards potential commercialization, planar platforms have proven to be inadequate to meet large‐scale manufacturing demand. Therefore, a new scalable platform for culturing anchorage‐dependent cells at high cell volumetric concentrations is urgently needed. One promising solution is to grow cells on microcarriers suspended in single‐use bioreactors. Toward this goal, a novel bioreactor system utilizing an innovative Vertical‐Wheel? technology was evaluated for its potential to support scalable cell culture process development. Two anchorage‐dependent human cell types were used: human lung carcinoma cells (A549 cell line) and human bone marrow‐derived mesenchymal stem cells (hMSC). Key hydrodynamic parameters such as power input, mixing time, Kolmogorov length scale, and shear stress were estimated. The performance of Vertical‐Wheel bioreactors (PBS‐VW) was then evaluated for A549 cell growth and oncolytic adenovirus type 5 production as well as for hMSC expansion. Regarding the first cell model, higher cell growth and number of infectious viruses per cell were achieved when compared with stirred tank (ST) bioreactors. For the hMSC model, although higher percentages of proliferative cells could be reached in the PBS‐VW compared with ST bioreactors, no significant differences in the cell volumetric concentration and expansion factor were observed. Noteworthy, the hMSC population generated in the PBS‐VW showed a significantly lower percentage of apoptotic cells as well as reduced levels of HLA‐DR positive cells. Overall, these results showed that process transfer from ST bioreactor to PBS‐VW, and scale‐up was successfully carried out for two different microcarrier‐based cell cultures. Ultimately, the data herein generated demonstrate the potential of Vertical‐Wheel bioreactors as a new scalable biomanufacturing platform for microcarrier‐based cell cultures of complex biopharmaceuticals. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1600–1612, 2015