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.     

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.     

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.     

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.     

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.     

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     

Cultivation of Thalictrum rugosum cell suspension in an improved airlift bioreactor: stimulatory effect of carbon dioxide and ethylene on alkaloid production

Airlift bioreactor operations have been studied for the growth-associated production of secondary metabolites from plant cell suspension cultures. The model system used in this work was Thalictrum rugosum producing berberine, an isoquinoline alkaloid. The airlift system was well suited for growth of Thalictrum cell suspension cultures unless the cell density was high. At high cell density, the airlift system with a draught tube was not adequate due to large aggregates clogging the recirculation paths. This was overcome by use of a cell scraper in the reactor. For berberine production, gas-stripping also played a significant role and it was discovered that CO(2) and ethylene were important for product formation. By supplying a mixture of CO(2) and ethylene into the airlift system, the specific berberine content was increased twofold. It is evident that continuous gas sparging was harmful for the production of berberine without supplementation with other gases.     

Alkaloid production in Catharanthus roseus cell cultures: Isolation and characterization of alkaloids from one cell line

A detailed study of one cell line (coded as 943) of Catharanthus roseus cell cultures has revealed the presence of the following alkaloids: ajmalicine, vallesiachotamine, hörhammerinine, hörhammericine, vindolinine, 19-epi- vindolinine, 19-acetoxy-11-methoxytabersonine, 19-acetoxy-11 -hydroxytabersonine, 19-hydroxy- 11-methoxytabersonine, yohimbine and isositsirikine, together with dimethyltryptamine and a new strychnos-type alkaloid.     

Development of a pheasant interspecies primordial germ cell transfer to chicken embryo: Effect of donor cell sex on chimeric semen production

This study was conducted to evaluate whether the sex of donor primordial germ cells (PGCs) influences production of chimeric semen from recipient hatchlings produced by interspecies transfer between pheasant (Phasianus colchicus) and chicken (Gallus gallus). Pheasant PGCs were retrieved from 7-d-old embryos and subsequently transferred into circulatory blood of 2.5-d-old (Stage 17) embryos. The sex of embryos was discerned 3 to 6 days after laying, and in preliminary study, overall rate of embryo survival after sexing was 74.6% with male-to-female ratio of 0.49 to 0.51. In Experiment 1, magnetic-activated cell sorting (MACS) using QCR1 antibody was effective for enriching the population of male and female PGCs in gonadal cells (9.2- to 12.5-fold and 10.8- to 19.5-fold increase, respectively). In Experiment 2, an increase in the number of hatchlings producing chimeric semen was detected after the homosexual transfer of male-to-male compared with that after the heterosexual transfer of female-to-male (68% to 88%). Significant increase was found in the frequency of chimeric semen production (0.96 to 1.68 times); production of pheasant progenies by artificial insemination using chimeric semen was also increased in the homosexual transfer (0 to 3 cases). In conclusion, the homosexual PGC transfer of male-to-male yielded better rate of generating pheasant progenies after test cross-reproduction than that of the heterosexual transfer of female-to-male, which could improve the efficiency of interspecies germ cell transfer system.     

High-density cultivation of Perilla frutescens cell suspensions for anthocyanin production: Effects of sucrose concentration and inoculum size

High-density cultivation of Perilla frutescens cells for anthocyanin production was carried out in both batch and fed-batch modes in a 500-ml shake flask. In fed-batch cultures, a high cell density of 27.7 g dry cells l⁻¹ and a total anthocyanin production of 3.87 g l⁻¹ by intermittent feeding of all medium components except hormones were obtained. In batch cultures, both initial sucrose concentration and inoculum size showed a conspicuous effect on the kinetics of cell growth, sugar consumption, and secondary metabolite (anthocyanins) production by suspended P. frutescens cells. At an inoculum size of 50 g wet cells l⁻¹, the maximum cell density of 38.3 g dry cells l⁻¹ was obtained after 11 days of cultivation at an initial sucrose concentration of 60 g l⁻¹, the highest pigment production of>5.8 g l⁻¹ was attained after 10 days of cultivation at an initial sucrose concentration of 45 g l⁻¹. These amounts of cell mass and anthocyanin pigments were 3.3 and 24 times higher than those at an initial sucrose concentration of 15 g l⁻¹ and inoculum size of 15 g wet cells l⁻¹, respectively.     

Biochemical properties of xylanases from a thermophilic fungus,Melanocarpus albomyces, and their action on plant cell walls

Melanocarpus albomyces, a thermophilic fungus isolated from compost by enrichment culture in a liquid medium containing sugarcane bagasse, produced cellulase-free xylanase in culture medium. The fungus was unusual in that xylanase activity was inducible not only by hemicellulosic material but also by the monomeric pentosan unit of xylan but not by glucose. Concentration of bagasse-grown culture filtrate protein followed by size-exclusion and anion-exchange chromatography separated four xylanase activities. Under identical conditions of protein purification, xylanase I was absent in the xylose-grown culture filtrate. Two xylanase activities, a minor xylanase IA and a major xylanase IIIA, were purified to apparent homogeneity from bagasse-grown cultures. Both xylanases were specific forβ-1,4 xylose-rich polymer, optimally active, respectively, at pH 6.6 and 5.6, and at 65°C. The xylanases were stable between pH 5 to 10 at 50°C for 24 h. Xylanases released xylobiose, xylotriose and higher oligomers from xylans from different sources. Xylanase IA had a M_r of 38 kDa and contained 7% carbohydrate whereas xylanase IIIA had a M_r of 24 kDa and no detectable carbohydrate. The K_m for larchwood xylan (mg ml⁻¹) and V_max (μmol xylose min⁻¹ mg⁻¹ protein) of xylanase IA were 0.33 and 311, and of xylanase IIIA 1.69 and 500, respectively. Xylanases IA, II and IIIA showed no synergism in the hydrolysis of larchwood glucuronoxylan or oat spelt and sugarcane bagasse arabinoxylans. They had different reactivity on untreated and delignified bagasse. The xylanases were more reactive than cellulase on delignified bagasse. Simultaneous treatment of delignified bagasse by xylanase and cellulase released more sugar than individual enzyme treatments. By contrast, the primary cell walls of a plant, particularly from the region of elongation, were more susceptible to the action of cellulase than xylanase. The effects of xylanase and cellulase on plant cell walls were consistent with the view that hemicellulose surrounds cellulose in plant cell walls.     

Effects of inbreeding and pollen donor provenance and diversity on offspring performance under environmental stress in the rare plant Cochlearia bavarica

Habitat degradation and loss can reduce size and genetic variability of natural populations, increasing individual homozygosity and average relatedness between individuals. While the resulting inbreeding depression may be reduced by natural selection under prevailing environmental conditions, it may increase again under environmental stress. To investigate the effect of environmental stress on offspring performance and the expression of inbreeding depression, we hand-pollinated maternal plants in small (< 100, n=5) and large populations (> 400 flowering plants, n=5) of the rare plant Cochlearia bavarica (Brassicaceae) and raised the offspring under experimentally manipulated water and light regimes (normal or reduced supply). In addition to considering natural variation in inbreeding levels due to population size, we manipulated pollen donor provenance and diversity. Maternal plants were pollinated with nine donors from a different population or with one or nine donors from the same population. One further inflorescence of each maternal plant was exposed to free pollination. Offspring growth and survival were monitored over 300 days. Offspring performance varied significantly among populations and maternal plants. Environmental stress interacted significantly with these factors. However, there was no general indication that offspring from small populations were more negatively affected. In seven out of 10 populations, offspring derived from between-population pollination performed better than offspring derived from within-population pollination. Also, in five out of 10 populations, average offspring size was higher after within-population pollination with nine than after pollination with one pollen donor. These results suggest low genetic diversity within C. bavarica populations, both smaller and larger ones. Interactions between environmental stress and pollination treatment indicated that using pollen donors from outside a population or increasing the number of pollen donors can reduce inbreeding depression, but that this beneficial effect is impaired under stressful conditions.     

Effects of dissolved oxygen tension and agitation rate on the production of heat-shock protein glycoprotein 96 by MethA tumor cell suspension culture in stirred-tank bioreactors

Heat-shock protein glycoprotein (gp96) serves as a natural adjuvant for chaperoning antigenic peptide into the immune surveillance pathway. In our laboratory, MethA tumor cell suspension culture process has been recently developed for gp96 production in spinner flask. In this work, effects of dissolved oxygen tension (DOT) and agitation rate on this process were studied in stirred-tank bioreactor. The optimal conditions for gp96 production were different with those for MethA tumor cell growth. MethA tumor cell growth pattern was not much changed by various levels of DOT and agitation rate, while gp96 biosynthesis was more sensitive to DOT and agitation rate. Compared with 50% of DOT, the production and specific productivity of gp96 was increased by 27 and 66% at 10% of DOT, respectively. Compared with the agitation rate of 100 rpm, the production and volumetric productivity of gp96 was increased by 48 and 144% at the agitation rate of 200 rpm, respectively. Low DOT (i.e., 10% of air saturation) and high agitation rate (i.e., 200 rpm) were identified to be favorable for gp96 biosynthesis. The results of this work might be useful to scale-up the bioprocess into the pilot scale.     

Effects of plant nutrient availability and host plant species on the performance of two Pieris butterflies (Lepidoptera: Pieridae)

We assayed the interaction on the availability of plant nutrient and species of host plant on the performance of two species of Pieris butterfly. The results indicated that constant application of different levels of fertilizers to the four different host plants resulted to an increase in their content of plant nutrients. The chemical analysis showed that the added nutrients increased foliar nitrogen and water contents, but there was no effect on the level of glucosinolates. Larvae that fed on highly-nutritious foliage increased their growth rates and showed a shorter development period. The results of feeding trials revealed that the 4th-instar larvae, which had fed on host plants with higher levels of fertilization had a shorter duration of development, less consumption rate, higher growth rate and food processing efficiency. To summarize, this research revealed that both the availability of plant nutrient and species of host plant can strongly influence the physiology and foliar chemistry of host plants. Moreover, the changes of phytochemical in the host plants may play an important role in affecting the performance (growth and food utilization efficiency) of both species of Pieris butterflies.     

Effects of TFAR19 gene on the in vivo biorheological properties and pathogenicity of mouse erythroleukemia cell line MEL

After injecting VP16, MEL cells and MEL-TF19 cells into the body of mice, with those injected with the same dose of saline as the control group, we observed the mice for their blood pictures, histological changes of the liver and spleen, and the hemorhelogical indexes within 4 weeks. The results indicated that after injecting MEL cells, the mice entered into a pathological status similar to erythroleukemia, which had the following exhibitions: the tissue structures of the liver and spleen were damaged, a mass of proerythroblasts, basophil erythroblasts and polychromatophilic erythroblasts could be observed on the smears of the bone marrow and spleen, and the deformability and orientation ability of erythrocytes were both depressed. The pathogenicity of MEL-TF19 cells carrying TFAR19 gene was obviously lower than that of MEL cells, and the MEL-TF19 cells even lost their faintish pathogenicity under the apoptosis-inducing effect of the chemotherapeutic reagent. The outcome from the animal experiments suggests that the TFAR19 gene suppresses the pathogenicity of MEL cells to the mice, and the effect may be better exerted with the synergy of the chemotherapeutic reagent. Supported by the National Natural Science Foundation of China (Grant Nos. 30270355 & 10572007) The first two authors should be regarded as joint first authors