Ultrasonic cell separator as a cell retaining device for high density cultures of plant cell

A system of ultrasonic filter device consisted of an ultrasonic generator, ultrasonic cell separation chamber (resonator) and a guide column, which was developed for suspension cultures of a plant cell. The key operation parameters affecting the efficiency of separation of cells from medium fluid were found to be the voltage of ultrasonic generator, the convective flow rate, and the distance between transducer and reflector. In the high density cultures ofAloe saponaria (>17 g DCW/L), the ultrasonic filter was so efficient that the cell holding time in the separation chamber was 10-fold higher than the case without ultrasonic wave at a convective flow rate of 0.24 cm/min. Furthermore, in perfusion type of high cell density cultures, cell aggregates were observed to be densely held in the ultrasonic chamber by ultrasonic force overcoming both gravitational and drag forces by pump. The accumulated cells were finally overflowed after the holding capacity of the chamber was reached. Back pressure was applied periodically to the resonator to flush cells back to bioreactor. The ultrasonic cell separator could operate over 75 min at a convective flow rate of 0.1 cm/min and at a cell concentration of 17 g DCW/L.     

Use of fed-batch cultivation for achieving high cell densities for the pilot-scale production of a recombinant protein (phenylalanine dehydrogenase) in Escherichia coli

A fed-batch process for the high cell density cultivation of E. coli TG1 and the production of the recombinant protein phenylalanine dehydrogenase (PheDH) was developed. A model based on Monod kinetics with overflow metabolism and incorporating acetate utilization kinetics was used to generate simulations that describe cell growth, acetate production and reconsumption, and glucose consumption during fed-batch cultivation. Using these simulations a predetermined feeding profile was elaborated that would maintain carbon-limited growth at a growth rate below the critical growth rate for acetate formation (mu < mu(crit)). Two starvation periods are incorporated into the feed profile in order to induce acetate utilization. Cell concentrations of 53 g dry cell weight (DCW)/L were obtained with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the total cell protein. The yield of PheDH was 129 U/mL with a specific activity of 1.2 U/mg DCW and a maximum product formation rate of 0.41 U/mg DCW x h. The concentration of aectate was maintained below growth inhibitory levels until 3 h before the end of the fermentation when the concentration reached a maximum of 10.7 g/L due to IPTG induction of the recombinant protein.     

Production of a platelet aggregation inhibitor, salmosin, by high cell density fermentation of recombinant Escherichia coli

Optimal conditions for a high cell density fermentation were investigated in a recombinant Escherichia coli producing salmosin, a platelet aggregation inhibitor. The optimized carbon and nitrogen sources were glycerol 10 g/l, yeast extract 30 g/l, and bacto-tryptone 10 g/l, yielding the dry cell weight (DCW) of 10.61 g/l in a 500 ml flask culture. The late-stage induction with 1% L-arabinose in a 5 l jar fermentor showed the highest DCW of 65.70 g/l after 27 h of the fed-batch fermentation. Around 2,200 mg/l of the protein was expressed as an inclusion body that was then refolded to obtain the active salmosin of 96 mg/l. We also confirmed the inhibitory activity against platelet aggregation of the active salmosin from the high cell density fermentation.     

Recombinant trypsin production in high cell density fed-batch cultures in Escherichia coli

Fed-batch techniques were employed to obtain high cell density cultures (92-100 g DCW/L) of Escherichia coli strain X90 producing a recombinant serine protease, rat anionic trypsin, secreted to the periplasm. The specific growth rate was controlled to minimize growth-inhibiting acetate formation by utilizing an exponential feeding profile determined from mass balance equation. The volumetric yield of recombinant rat anionic trypsin was 56 mg/L, and the final cell density was 92 g DCW/L when the culture was induced in the late logarithmic phase. However, when the culture was induced in the early logarithmic phase, the volumetric yield was 13 mg/L and the final cell density was 14 g DCW/L. Thus, the induction timing is shown to have a significant effect on the final cell density as well as the overall volumetric yield of the recombinant protease. (c) 1993 Wiley & Sons, Inc.     

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.     

Changes in buoyant density and cell size of Escherichia coli in response to osmotic shocks.

The buoyant density of Escherichia coli was shown to be related to the osmolarity of the growth medium. This was true whether the osmolarity was adjusted with either NaCl or sucrose. When cells were grown at one osmolarity and shocked to another osmolarity, their buoyant density adjusted to nearly suit the new osmolarity. When cells were subjected to hyperosmotic shock, they became denser than expected. When cells were subjected to hypoosmotic shock they occasionally undershot the new projected density, but the undershoot was not as dramatic as the overshoot seen with hyperosmotic shocks. Shrinkage and swelling of the cells in response to osmotic shocks could account for the change in their buoyant density. The changes in cell size after osmotic shocks were measured by two independent methods. The first method measured cell size with a Coulter Counter, and the second method measured cell size by stereologic analysis of Nomarski light micrographs. Both methods gave qualitatively similar results and showed the cells to be flexible. The maximum swelling recorded was 23% of the original cell volume, while the maximum shrinkage observed was 33%.     

Expanded bed adsorption of human serum albumin from very dense Saccharomyces cerevesiae suspensions on fluoride-modified zirconia.

The adsorption of proteins from high cell density yeast suspensions on mixed-mode fluoride-modified zirconia (FmZr) particles (38 to 75 microm, surface area of 29 m(2)/g and density of 2.8 g/cm(3)) was investigated using human serum albumin (HSA) added to Saccharomyces cerevesiae as the model expression host. Because of the high density of the porous zirconia particles, HSA (4 mg/mL) can be adsorbed from a 100 g dry cell weight (DCW)/L yeast suspension in a threefold-expanded bed of FmZr. The expanded bed adsorption of any protein from a suspension containing >50 g DCW/L cells has not been previously reported. The FmZr bed expansion characteristics were well represented by the Richardson-Zaki correlation with a particle terminal velocity of 3.1 mm/s and a bed expansion index of 5.4. Expanded bed hydrodynamics were investigated as a function of bed expansion using residence time distribution studies with sodium nitrite as the tracer. The adsorption of HSA on FmZr exhibited features of multicomponent adsorption due to the presence of dimers. The protein binding capacity at 5% breakthrough decreased from 22 mg HSA/mL settled bed void volume for 20 g DCW/L yeast to 15 mg HSA/mL settled bed void volume for 40 g DCW/L yeast and remained unchanged for the higher yeast concentrations (60 to 100 g DCW/L). However, the batch (or equilibrium) binding capacity decreased monotonically as a function of yeast concentration (20 to 100 g DCW/L) and the binding capacity at 100 g DCW/L yeast was fivefold lower compared with that at 20 g DCW/L yeast. The lower batch binding capacity at high cell concentrations resulted from the adsorption of cells at the surface of the particles restricting access of HSA to the intraparticle surface area. Batch (or equilibrium) and column HSA adsorption results indicated that the adsorption of HSA on FmZr occurred at a time scale that may be much faster than that of yeast cells. The zirconia particles were cleaned of adsorbed HSA and yeast with a total of 1500 to 2000 column volumes (over many cycles) of 0. 25 M NaOH, without any significant effect on the chromatographic performance.     

Induction of lignans and phenolic compounds in cell culture of Linum album by culture filtrate of Fusarium graminearum

Cell suspension cultures of Linum album Kotschy ex Boiss. have been reported to produce anticancer podophyllotoxin and its related lignans. In the present study, we investigated the effect of culture filtrate of Fusarium graminearumon growth, and lignan and phenolic compounds in L. album cell culture. After 7 days of pre-culture, the cells were treated with 1% (v/v) of the culture filtrate. Cell growth was reduced, while phodophyllotoxin and lariciresinol production was stimulated reaching a maximum 0.0187 mg/g fresh weight (FW) and 0.0136 mg/g FW 5 days after the treatment, respectively. Also, our results provide evidence that the culture filtrate of F. graminearum can be effective on phenylalanine ammonia-lyase activity and phenolic compounds.     

A new fermentation strategy for S-adenosylmethionine production in recombinant Pichia pastoris

A recombinant Pichia pastoris MutS expressing SAM2 gene of Saccharomyces cerevisiae was cultured for S-adenosylmethionine (SAM) accumulation. Effect of the amount of methanol added (0.5%, 1.0%, 2.0%, 3.0%, 4.0%, 6.0%, 10.0%, and 12.0%) and cell densities (9.57, 13.47, 21.74, 30.90, and 41.24 g/L dry cell weight (DCW)) on yield of SAM was found in flask cultivations. In flask experiments, maximal yield of SAM (1.29 g/L) was obtained at 2.0% methanol added and 30.90 g/L DCW which gave the maximal methanol consumption rate. Conjunct effect of amount of methanol added and cell density was found through Origin 7.0 (7.0 Microcal, USA). Scale up in 3.7 L bioreactor, 51% specific yield of SAM was enhanced at 0.6% methanol compared to that of 0.1% methanol. In fed-batches of different cell densities at 0.6% methanol, maximal yield of SAM was 8.66 g/L at 100 g/L DCW with 64% yield of SAM enhanced again. Methanol consumption rate at 100 g/L DCW was 4.81 mL/L h. Maintenance coefficient of 100 g/L DCW was lower than that of others significantly, although methanol consumption rate of 90 g/L DCW was higher (5.07 mL/L h) than that of 100 g/L DCW.     

Simple fed-batch technique for high cell density cultivation of Escherichia coli

A simple fed-batch process for high cell density cultivation of Escherichia coli TG1 was developed. A pre-determined feeding strategy was chosen to maintain carbon-limited growth using a defined medium. Feeding was carried out to increase the cell mass concentration exponentially in the bioreactor controlling biomass accumulation at growth rates which do not cause the formation of acetic acid (μ < μ_crit). Cell concentrations of 128 and 148 g per 1 dry cell weight (g 1⁻¹ DCW) were obtained using glucose or glycerol as carbon source, respectively.     

碱性果胶酶在重组毕赤酵母中高效表达的关键因素研究

为提高重组毕赤酵母生产碱性果胶酶的产量和生产强度, 在摇瓶条件下优化了重组毕赤酵母生产碱性果胶酶的关键因素。结果表明, 以下条件：初始甘油浓度40 g/L、初始甲醇浓度3.1 g甲醇/g DCW、每24 h添加0.51 g甲醇/g DCW、诱导表达周期72 h、250 mL三角瓶诱导培养基装液量30 mL、初始pH 6.0, 最适于菌体生长与产物表达。在此基础上, 7 L罐上通过恒速流加甘油进一步提高细胞密度, 诱导阶段甲醇采取前期恒速流加和后期DO-stat, 发酵结束菌体干重达80 g/L, 酶活为217 U/mL, 比摇瓶结果提高了66.2%。     

碱性果胶酶在重组毕赤酵母中高效表达的关键因素研究

为提高重组毕赤酵母生产碱性果胶酶的产量和生产强度,在摇瓶条件下优化了重组毕赤酵母生产碱性果胶酶的关键因素。结果表明,以下条件：初始甘油浓度40g／L、初始甲醇浓度3.1g甲醇／gDCW、每24h添加0.51g甲醇／gDCW、诱导表达周期72h、250mL三角瓶诱导培养基装液量30mL、初始pH6,0,最适于菌体生长与产物表达。在此基础上,7L罐上通过恒速流加甘油进一步提高细胞密度,诱导阶段甲醇采取前期恒速流加和后期DO-stat,发酵结束菌体干重达80g／L,酶活为217U／mL,比摇瓶结果提高了66.2％。     

Cell morphometry and osmolarity as early indicators of the onset of embryogenesis from cell suspension cultures of grain legumes and model systems

Abstract

Whatever the in vitro regeneration pathway, it would be of interest to be able to distinguish regeneration-competent from non-regenerating cells and tissues, and as early in culture as possible, as this would allow a dramatic improvement of biotechnology breeding, particularly for the so-called recalcitrant species. With this aim, we examined a range of genotypes of pea (Pisum sativum) and grass pea (Lathyrus sativus), and of the two model species Medicago truncatula, another legume, and Arabidopsis thaliana. This was done by comparing cell suspension cultures of different ages (young [<6 transfers-old]vs. mature [>6 transfers-old]), densities (dense [>10⁷ cells/ml] or sparse [<10⁶ cells/ml]) and regeneration abilities (non-embryogenic vs. embryogenic), in order to identify early indicators of competence for somatic embryogenesis. All such cell suspensions were subcultured every 14 days and several parameters were assessed every 3–4 days during each 14-day cycle. These included the time course pH and osmolarity of the culture medium, the internal osmolarity of cells, the cell surface and the cell wall thickness (by examining cellulose accumulation in Calcofluor White-stained cells under UV light). As cells underwent embryogenesis they enlarged. Cellulose accumulated in the walls of non-embryogenic cells, but walls became thinner with the onset of embryogenesis, and diminished further as embryos matured. Although medium osmolarity decreased at the onset of embryogenesis, this was never observed for non-embryogenic cell suspensions. Conversely, there was a concomitant increase in intracellular osmolarity for embryogenic cells. Medium pH (analysed with the model species only) was not significantly correlated with regeneration competence of cells. For all genotypes and species, the kinetics of cell wall thickness and cell surface, and that of medium and cell osmolarity were reliable early indicators of the competence of cells to undergo somatic embryogenesis. The implication of these results for biotechnological breeding of grain legumes and for plant regeneration competence in general are discussed.     

Novel electrochemical-enzymatic model which quantifies the effect of the solution Eh on the kinetics of ferrous iron oxidation with Acidithiobacillus ferrooxidans

The heterologous production of epothilone D in Myxococcus xanthus was improved by 140-fold from an initial titer of 0.16 mg/L with the incorporation of an adsorber resin, the identification of a suitable carbon source, and the implementation of a fed-batch process. To reduce the degradation of epothilone D in the basal medium, XAD-16 (20 g/L) was added to stabilize the secreted product. This greatly facilitated its recovery and enhanced the yield by three-fold. The potential of using oils as a carbon source for cell growth and product formation was also evaluated. From a screen of various oils, methyl oleate was shown to have the greatest impact. At the optimal concentration of 7 mL/L in a batch process, the maximum cell density was increased from 0.4 g dry cell weight (DCW)/L to 2 g DCW/L. Product yield, however, depended on the presence of trace elements in the production medium. With an exogenous supplement of trace metals to the basal medium, the peak epothilone D titer was enhanced eight-fold. This finding demonstrates the significant role of metal ions in cell metabolism and in epothilone biosynthesis. To further increase the product yield, a continuous fed-batch process was used to promote a higher cell density and to maintain an extended production period. The optimized fed-batch cultures consistently yielded a cell density of 7 g DCW/L and an average production titer of 23 mg/L.     

Bioreactor seaweed cell culture for production of bioactive oxylipins

Liquid cell suspension cultures derived from marine plants have the potential to biosynthesize novel biomedicinal compounds in a controlled environment. Of particular interest are the eicosanoids and related oxylipins emanating from the 15-lipoxygenase manifold of the arachidonic acid cascade, which is active in the brown algaLaminaria saccharina. Filamentous cell clumps ofL. saccharina isolated from female gametophytes were cultured in an illuminated bubble-column bioreactor in GP2 artificial seawater nutrient medium at 13 °C and air flow rate of 0.35 L air min^–1 L^–1 culture (vvm). Growth kinetics and biomass productivity data were obtained as a function of incident light intensity (2.4 to 98mol photon m^–2 s^–1) and initial cell density (27 to 149 mg DCW L^–1). Maximum cell densities exceeded 1200 mg DCW L^–1 after a 20 day cultivation time at optimal conditions of 98mol photon m^–2 s^–1 and 118 mg DCW L^–1 initial cell density. Qualitative analysis of chloroform/methanol extracts of the cell culture biomass by GC-MS confirmed the presence of the hydroxy fatty acids 13-HODTA and 13-HOTE, the likely products of 15-lipoxygenase catalyzed oxidation of linoleic or linolenic acids.     

Effects of calcium,alginate, and calcium-alginate immobilization on growth and tropane alkaloid levels of a stable suspension cell line of Datura innoxia Mill

Summary A stabilized two-year old suspension of a Datura innoxia cell line, producing small amounts of tropane alkaloids (scopolamine and hyoscyamine) was used in this study. Calcium alginate immobilization has been shown to be able to increase secondary metabolite (i. e. alkaloid) production. The effects of calcium and ungellified alginate were both beneficial for tropane alkaloid synthesis; a 10mM calcium chloride supply gave the best results, with a 10-fold yield increase.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - S standard cell culture medium - HPLC high performance liquid chromatography - FW fresh weight - FDA fluorescein diacetate - FW fresh weight     

重组巴氏毕赤酵母恒化培养动力学及代谢迁移特性研究

通过对甲醇营养型毕赤酵母基因工程菌以碳源甘油为限制性基质进行恒化培养动力学试验 ,结果认为 :(1 )细胞光密度与其干、湿重呈线性关系 ,当细胞光密度 (OD60 0 )为 1 0 0时细胞湿重 (WCW)为 1 2 8 3g L ,细胞干重 (WDW)则为 2 2 9g L ;(2 )基因工程菌P .pastoris的生长与限制性基质甘油残留浓度的关系符合Monod关系式 ,通过 1 μ对 1 S进行线性回归得 μmax=0 .366h- 1,Ks=0 .1 82 3g L ,经参数推导甘油最大菌体得率系数YG =0 .54g g ,菌体维持生长消耗底物系数m =0 .0 0 69g (g·h) ;氧最大菌体系数YX O2 =30 .96g moL ,菌体维持生长时消耗氧系数mO2 =0 .0 0 0 8mol (g·h) ,最适理论稀释速率Dm =0 .341h- 1;(3)从氨水的消耗速率和呼吸商 (RQ)的变化认为随着比生长速率 (μ)的增大 ,甘油代谢流从糖原异生和磷酸戊糖途径线性地向糖酵解和三羧酸循环途径进行代谢迁移 ,即糖酵解和三羧酸循环途径的代谢流量在线性地增大     

Enhanced release of rosmarinic acid from Coleus blumei permeabilized by dimethyl sulfoxide (DMSO) while preserving cell viability and growth

Continuous permeabilization of preconditioned Coleus blumei cells with dimethyl sulfoxide (DMSO) is shown to be an effective strategy for the enhanced release of rosmarinic acid (RA) while preserving cell viability. When nonpreconditioned cells were permeabilized with DMSO, they lost their viability at DMSO concentrations higher than a critical value located between 0.1% and 0.5% DMSO. Product release was low [0.49 g RA/100 g dry cell weight (DCW)] at 0.1% DMSO. Preconditioning cells at 0.1% DMSO ensured high viability at DMSO concentrations of 0.5%, 1.0%, and 1.5%. Product release reached a maximum of 2.85 g RA/100 g DCW at 0.5% DMSO, which was 66.4% of the total rosmarinic acid produced. (c) 1992 John Wiley & Sons, Inc.     

Identification and production of flavonoids in a cell suspension culture of Scutellaria baicalensis G.

A high yielding cell line of Scutellaria baicalensis G. has successfully been developed to produce flavonoids. Major components of the flavonoids were identified as baicalin and wogonin-7-O-glucuronic acid by a series of instrumental analyses using UV, IR, MASS, and NMR. After 12 days in suspension culture, the cell growth reached 14 g ^DW/l, and baicalin and wogonin-7-O-glucuronic acid were obtained in concentrations of 2.9 g/l and 1.07 g/l, respectively. The culture temperature was found to be an important parameter for improving production yield of the flavonoids. The yield of baicalin was observed to increase to 4.2 g/l by shifting the temperature from 30 °C to 25 °C after 72 h of suspension culture.Abbreviations DW cell dry weight - FW cell fresh weight - 2,4-D 2,4-dichlorophenoxyacetic acid - PSH medium phytohormone added Schenk and Hildebrandt medium - FPM a modified Schenk and Hildebrandt medium for flavonoid production