Dynamics of oxygen evolution and biomass production during cultivation of Agardhiella subulata microplantlets in a bubble-column photobioreactor under medium perfusion

Cell and tissue cultures derived from macrophytic marine red algae are potential platforms for unique secondary metabolites. This work presents the first successful bioreactor cultivation study of an in vitro tissue culture derived from a macrophytic marine red alga. Specifically, the photosynthetic growth characteristics of a novel microplantlet suspension culture established from the macrophytic marine red alga Agardhiella subulata were studied. A bubble-column bioreactor with external illumination (43 microE m(-2) s(-1), 10:14 LD photoperiod), liquid medium perfusion, and 3800 ppm CO(2) in the aeration gas provided sufficient light and nutrient delivery for sustained growth of the microplantlet suspension at 24 degrees C and pH 8. Microplantlets, which consisted of shoot tissues of 3-5 mm length branching out from a common center, were not friable in a bubble-aerated suspension of about 1100 plantlets per liter. Since the microplantlet tissues were not friable, only batch and fed-batch cultivation modes were considered. Batch cultivation was phosphate-limited in ASP12 artificial seawater medium. However, cultivation at a medium perfusion rate of 20% per day avoided phosphate limitation and extended the growth phase to provide plantlet mass densities exceeding 14 g FW L(-1) (3.7 g DW L(-1)) after 50 days of cultivation if the suspension was not sampled. The specific oxygen evolution rate vs cultivation time profile possessed a significant pulse within the 14 days following inoculation and then leveled off at longer times. In recognition of this nonexponential growth pattern, a new photobioreactor growth model was developed that used the oxygen evolution rate vs time profile to predict the biomass growth curve in perfusion culture. Model predictions agreed reasonably with the measured growth curves.     

Monoterpenoid oxindole alkaloid production by Uncaria tomentosa (Willd) D.C. cell suspension cultures in a stirred tank bioreactor

Cell growth, monoterpenoid oxindole alkaloid (MOA) production, and morphological properties of Uncaria tomentosa cell suspension cultures in a 2-L stirred tank bioreactor were investigated. U. tomentosa (cell line green Uth-3) was able to grow in a stirred tank at an impeller tip speed of 95 cm/s (agitation speed of 400 rpm), showing a maximum biomass yield of 11.9 +/- 0.6 g DW/L and a specific growth rate of 0.102 d(-1). U. tomentosa cells growing in a stirred tank achieved maximum volumetric and specific MOA concentration (467.7 +/- 40.0 microg/L, 44.6 +/- 5.2 microg/g DW) at 16 days of culture. MOA chemical profile of cell suspension cultures growing in a stirred tank resembled that of the plant. Depending on culture time, from the total MOA produced, 37-100% was found in the medium in the bioreactor culture. MOA concentration achieved in a stirred tank was up to 10-fold higher than that obtained in Erlenmeyer flasks (agitated at 110 rpm). In a stirred tank, average area of the single cells of U. tomentosa increased up to 4-fold, and elliptical form factor increased from 1.40 to 2.55, indicating enlargement of U. tomentosa single cells. This work presents the first report of U. tomentosa green cell suspension cultures that grow and produce MOA in a stirred tank bioreactor.     

Halogenated monoterpene production by microplantlets of the marine red alga Ochtodes secundiramea within an airlift photobioreactor under nutrient medium perfusion

Macrophytic marine red algae are a unique source of novel and bioactive terpenoids, including halogenated monoterpenes. Biomass and halogenated monoterpene production by regenerated microplantlet suspension cultures derived from the red alga Ochtodes secundiramea were studied within a perfusion airlift photobioreactor. Photobioreactor cultivations were carried out at 26 degrees C, 140 microE m(-2)s(-1) light intensity, 0.3 air L(-1) culture min(-1) aeration (3500 ppm CO(2)), and ESS/seawater medium perfusion rate of 0.2 L medium L(-1) culture d(-1). Macronutrient concentrations in the perfusion medium were adjusted to provide nitrate delivery rates of 0.0063, 0.077, and 0.74 mmol L(-1) d(-1) at a fixed N:P ratio of 19:1. Growth was maximized at the highest nutrient delivery rate, where 10 g dry biomass L(-1) culture was achieved after 30 days of cultivation. GC-MS analysis of dichloromethane extracts from cell biomass revealed that O. secundiramea microplantlets produced myrcene, three acyclic halogenated monoterpenes (10-bromomyrcene, 10-bromo-7-chloromyrcene, 3,10-dibromomyrcene), and one cyclic halogenated monoterpene (6-bromo-1,2,8-trichloro-3,4-ochtodene). 10E-bromomyrcene levels were much higher than those of its isomer 10Z-bromomyrcene, demonstrating stereoselective halogenation. Maximum yields of 10E-bromomyrcene and 6-bromo-1,2,8-trichloro-3,4-ochtodene were 15 and 13 micromol/g dry cell mass, respectively. Increasing the rate of nutrient delivery increased the accumulation of myrcene and 10-bromomyrcene during the first 14 days in culture. Furthermore, the yield selectivity toward higher halogenated monoterpenes increased as the rate of nutrient delivery decreased. From this data, a biogenic scheme was proposed where cyclic and acyclic halogenated monoterpenes are derived from sequential halogenation of myrcene, their common precursor.     

Optimal temperature and photoperiod for the cultivation of Agardhiella subulata microplantlets in a bubble-column photobioreactor

The optimal temperature and illumination photoperiod requirements for the phototrophic growth of a novel microplantlet suspension culture derived from the macrophytic marine red alga Agardhiella subulata were determined. The optimal growth temperature was 24 degrees C. The effects of illumination light-dark (LD) photoperiod (hour of light:hours of darkness within a 24 h cycle) on biomass production was studied within a bubble-column photobioreactor. The 4.5 cm diameter photobioreactor was maintained at near-saturation conditions with respect to light flux (38 mciromol photons m(-2) s(-1)), nutrient medium delivery (20% nutrient replacement per day), and CO(2) delivery (0.35 mmol CO(2) L(-1) h(-1)) so that the cumulative effects of photodamage on the cell density versus time curve at photoperiods approaching continuous light could be observed. Biomass production was maximized at 16:8 LD, where biomass densities exceeding 3.6 g dry cell mass L(-1) were achieved after 60 days in culture. Biomass production was proportional to photoperiod at low fractional photoperiods (< or =10:14 LD), but high fractional photoperiods approaching continuous light (> or = 20:4 LD) shut down biomass production. Biomass production versus time profiles under resource-saturated cultivation conditions were adequately described by a cumulative photodamage growth model, which coupled reversible photodamage processes to the specific growth rate. Under light-saturated growth conditions, the rate constant for photodamage was kd = 1.17 +/- 0.28 day(-1) (+/-1.0 SE), and the rate constant for photodamage repair was kr = 5.12 +/- 0.95 day(-1) (+/-1.0 SE) at 24 degrees C.     

Development and bioreactor cultivation of a novel semidifferentiated tissue suspension derived from the marine plant Acrosiphonia coalita

A semidifferentiated tissue culture consisting of linear filaments in liquid suspension was established from Acrosiphonia coalita, a cold-water green macroalga known to express pharmacologically active oxylipins deriving from lipoxygenase metabolism of linolenic acid. The tissue was vegatively propagated by blending the filaments down to 1 to 5 mm in length prior to subculture. The filamentous A. coalita tissue suspension was successfully cultivated in an illuminated, 3-L stirred-tank bioreactor at 12 degrees C, 0.46-vvm aeration rate, 250-rpm mixing speed, and incident illumination intensity of 77 muE m(-2)s(-1). The mean specific growth rate over the exponential phase was 0.185 day(-1) and a final cell density of 1083 mg dry cell weight (DCW) L(-1) was achieved within 15 days of cultivation from an initial cell density of 200 mg DCW L(-1). The addition of 3500 ppm CO(2) to the aeration gas provided a maximum CO(2) transfer rate of six times the maximum CO(2) consumption rate and stabilized the pH to 8.0 during the light phase of growth, but did not improve biomass productivity. (c) 1996 John Wiley & Sons, Inc.     

Culture of insect cells in helical ribbon impeller bioreactor

An 11-L helical ribbon impeller (HRI) bioreactor was tested for the culture of Spodoptera frugiperda (Sf-9) cells. This impeller and surface baffling ensured homogeneous mixing and high oxygen transfer through surface aeration and surface-induced babble generation. Serum-supplemented and serum-free cultures, using TNMFH and IPL/41 media, respectively, grew a similar specific growth rates(0.031 and 0.028 h(-1)) to maximum cell densities of 5.5 x 10(6)-6.0 x 10(6) cells. mL(-1) with viability exceeding 98% during exponential growth phase. Growth limitation coincided with glucose and glutamine depletion and production of significant amounts of alanine. The bioreactor was further tested under more stringent conditions by infecting a serum-free medium culture with a recombinant baculovirus. Heterologous protein production of approximately 35 mug per 10(6) cells was comparable to yields obtained in serum-free cultures grown in spinner flasks and petri dishes. Average specific oxygen up-take and carbon dioxide production rates of the serum-free culture prior to infection as measured by on-line mass spectroscopy were 0.20 mumol O(2)mu.(10(6) cells)(-1) h(-1) and 0.22 mumol CO(2) . (10(6) cells)(-1)h(-1) and increased by 30-40% during infection. Therefore, the mixing and oxygenation conditions of this bioreactor were suitable for insect cell culture and recombinant protein production, with limitation being mainly attributed to nutrient depletion and toxic by-product generation.     

Effect of aeration and agitation on the protease production by S<Emphasis Type="Italic">taphylococcus aureus</Emphasis> mutant RC128 in a stirred tank bioreactor

The modified rotating simplex method has been successfully used to determine the best combination of agitation rate and aeration rate for maximum production of extracellular proteases by Staphylococcus aureus mutant RC128, in a stirred tank bioreactor operated in a discontinuous way. This mutant has shown altered exoprotein production, specially enhanced protease production. Maximum production of proteases (15.28 UP/ml), measured using azocasein as a substrate, was obtained at exponential growth phase when the bioreactor was operated at 300 rpm and at 2 vvm with a volumetric oxygen transfer coefficient (K _L a) of 175.75 h⁻¹. These conditions were found to be more suitable for protease production.     

Azadirachtin production in stirred tank reactors by Azadirachta indica suspension culture

Successful scale-up of Azadirachta indica suspension culture for azadirachtin production was done in stirred tank bioreactor with two different impellers. The kinetics of biomass accumulation, nutrient consumption and azadirachtin production of A. indica cell suspension culture were studied in a stirred tank bioreactor equipped with centrifugal impeller and compared with similar bioreactor with a setric impeller to investigate the role of O₂ transfer efficiency of centrifugal impeller bioreactor on overall culture metabolism. The maximum cell mass for centrifugal impeller bioreactor and stirred tank bioreactor (with setric impeller) were 18.7 and 15.5 g/L (by dry cell weight) and corresponding azadirachtin concentrations were 0.071 and 0.05 g/L, respectively. Glucose and phosphate were identified as the major growth-limiting nutrients during the bioreactor cultivation. The centrifugal impeller bioreactor demonstrated less shearing and improved O₂ transfer than the stirred tank bioreactor equipped with setric impeller with respect to biomass and azadirachtin production.     

甘草细胞放大培养中搅拌式反应器操作策略优化

为获得甘草细胞在反应器中放大培养的最佳条件,在建立稳定的甘草细胞搅拌式生物反应器放大培养体系的基础上,分别以单因素和正交实验获得的数据为样本,以细胞净增长生物量为考察指标,运用BP神经网络耦合遗传算法对反应器操作策略进行优化。结果表明,接种量6.4%、摇床转速89r/min、通气速率0.1vvm是甘草细胞进行反应器培养的最优条件;与传统的正交实验方法相比,这种基于神经网络耦合遗传算法的优化方法使反应器中细胞生物量的积累提高了6.9%。     

Effect of carbon source and aeration rate on broth rheology and fungal morphology during red pigment production by Paecilomyces sinclairii in a batch bioreactor

The influence of carbon source and aeration rate on fermentation broth rheology, mycelial morphology and red pigment production of Paecilomyces sinclairii was investigated in a 5-l stirred-tank bioreactor. The characteristics of P. sinclairii grown on starch and on sucrose medium were comparatively studied: the specific growth rate in sucrose medium (0.04 h(-1)) was higher than that in starch medium, whereas the specific production rate of red pigments (0.04 gg(-1)d(-1)) was favorable in starch medium. P. sinclairii grown in sucrose medium were highly branched and showed longer hyphal lengths than that in starch medium. The consistency index (K) in sucrose medium was markedly higher than that in starch medium due to higher cell mass, while the higher values of flow behavior index (n) were indicated at the late stationary phase in starch medium. The aeration rate was varied within the ranges from 0.5 to 3.5 vvm while running the fermentation at mild agitation of 150 rpm using sucrose as the carbon source. The maximum biomass concentration of P. sinclairii was about 33 gl(-1) with an aeration rate of 1.5 vvm, whereas the maximum yield of red pigment production (4.73 gl(-1)) was achieved with 3.5 vvm. The highly branched cell morphology appeared at 1.5 vvm and the highly vacuolated cell morphology was observed in a high aeration rate (3.5 vvm). There was no significant variance in rheological parameters (K and n) between culture broths from different aeration conditions.     

Scaleup of ajmalicine production by plant cell cultures of Catharanthus roseus

The effect of scaleup on he production of ajmalicine by a Catharanthus roseus cell suspension culture in a selected induction medium were studied. In preliminary experiments it was observed that the culture turned brown and the production was inhibited upon transfer from a shake flask to a stirred bioreactor with forced aeration. Two factors were recognized as the potential origin of the differences between shake flask and bioreactor cultures: gas composition and mechanical shear forces. These factors were studied separately.By recirculating a large part of the exhaust gas, a comparable gas regime was obtained in a bioreactor as occurred in a shake flask cultures. This resulted in the absence of browning and a similar pattern of ajmalicine production as observed in shake flasks. The effect of shear forces could not be demonstrated. However, the experiments showed that the culture may be very sensitive to liquid phase concentrations of gaseous compounds. The effects of k(L)a, aeration rate, CO(2) production rate, and influent gas phase CO(2) concentration on the liquid phase CO(2) concentration are discussed. (c) 1993 John Wiley & Sons, Inc.     

Short Communication: Effect of agitation rate on the growth and production of xylanase free of cellulase by Thermomyces lanuginosus MH4 in bioreactor

Cellulase-free xylanase production by T. lanuginosus MH4 was investigated in a 3-litre stirred tank bioreactor under different agitation rates and an aeration rate of 1v/v/m. The cultivation time in the bioreactor was reduced significantly over that in shake culture conditions. A xylanase productivity of 0.1 mkat1–1h–1 was achieved on xylan in the bioreactor. This was nearly double to that obtained in shake culture. The agitation rates influenced both growth and enzyme secretion in the bioreactor. The highest level of biomass concentration and activities of both xylanase and -xylosidase were obtained at 150 revmin–1     

Comparison of manufacturing techniques for adenovirus production

We have compared three different production methods, which may be suitable for the large scale production of adenovirus vectors for human clinical trials. The procedures compared 293 cells adapted to suspension growth in serum-free medium in a stirred tank bioreactor, 293 cells on microcarriers in serum-containing medium in a stirred tank bioreactor, and 293 cells grown in standard tissue culture plasticware. With a given virus, yields varied between 2000 and 10,000 infectious units/cell. The stirred tank bioreactor routinely produced between 4000 and 7000 infectious units/cell when 293 cells were grown on microcarriers. The 293 cells adapted to suspension growth in serum-free medium in the same stirred tank bioreactor yielded between 2000 and 7000 infectious units/cell. Yields obtained from standard tissue culture plasticware were up to 10,000 infectious units/cell. Cell culture conditions were monitored for glucose consumption, lactate production, and ammonia accumulation. Glucose consumption and lactate accumulation correlated well with the cell growth parameters. Ammonia production does not appear to be significant. Based on virus yields, ease of operation and linear scalability, large-scale adenovirus production seems feasible using 293 cells (adapted to suspension/serum free medium or on microcarriers in serum containing medium) in a stirred tank bioreactor. This revised version was published online in July 2006 with corrections to the Cover Date.     

High cell density cultivation of recombinant yeasts and bacteria under non-pressurized and pressurized conditions in stirred tank bioreactors

This study demonstrates the applicability of pressurized stirred tank bioreactors for oxygen transfer enhancement in aerobic cultivation processes. The specific power input and the reactor pressure was employed as process variable. As model organism Escherichia coli, Arxula adeninivorans, Saccharomyces cerevisiae and Corynebacterium glutamicum were cultivated to high cell densities. By applying specific power inputs of approx. 48kWm(-3) the oxygen transfer rate of a E. coli culture in the non-pressurized stirred tank bioreactor was lifted up to values of 0.51moll(-1)h(-1). When a reactor pressure up to 10bar was applied, the oxygen transfer rate of a pressurized stirred tank bioreactor was lifted up to values of 0.89moll(-1)h(-1). The non-pressurized stirred tank bioreactor was able to support non-oxygen limited growth of cell densities of more than 40gl(-1) cell dry weight (CDW) of E. coli, whereas the pressurized stirred tank bioreactor was able to support non-oxygen limited growth of cell densities up to 225gl(-1) CDW of A. adeninivorans, 89gl(-1) CDW of S. cerevisiae, 226gl(-1) CDW of C. glutamicum and 110gl(-1) CDW of E. coli. Compared to literature data, some of these cell densities are the highest values ever achieved in high cell density cultivation of microorganisms in stirred tank bioreactors. By comparing the specific power inputs as well as the k(L)a values of both systems, it is demonstrated that only the pressure is a scaleable tool for oxygen transfer enhancement in industrial stirred tank bioreactors. Furthermore, it was shown that increased carbon dioxide partial pressures did not remarkably inhibit the growth of the investigated model organisms.     

Production of carbonyl reductase by Metschnikowia koreensis

A new strain of the yeast Metschnikowia koreensis was grown in shake flasks and a stirred bioreactor for the production of carbonyl reductase. The optimal conditions in the bioreactor for maximizing the biomass specific activity of the enzyme were found to be: a medium composed of glucose (20 g/L), peptone (5 g/L), yeast extract (5 g/L) and zinc sulfate (0.3g/L); the pH controlled at 7; the temperature controlled at 25 °C; an agitation speed of 500 rpm; and an aeration rate of 0.25 vvm. In the bioreactor, a biomass specific enzyme activity of 115.6 U/gDCW was obtained and the maximum biomass concentration was 15.3 gDCW/L. The biomass specific enzyme activity obtained in the optimized bioreactor culture was 11-fold higher than the best result achieved in shake flasks. The bioreactor culture afforded a 2.7-fold higher biomass concentration than could be attained in shake flasks.     

Cultivation of transgenicNicotiana tabacum suspension cells in bioreactors for the production of mGM-CSF

TransgenicNicotiana tabacum cells were cultivated for the production of murine granulocyte macrophage-colony stimulating factor (mGM-CSF) in both a stirred, tank biore|actor and an airlift bioreactor with draft tube. Cell growth and mGM-CSF production in the airlift bioreactor were found to be better than those achieved in the stirred tank bioreactor. In the airlift bioreactor. 9.0 g/L of cells and 2.2 ng/mL of mGM-CSF were obtained (11.0 g/L and 2.4 ng/mL, respectively in shake flasks). Although the lag period was prolonged and mGM-CSF production was lowered by 33% in the stirred tank bioreactor as compared to the control culture, the maximum cell density was increased up to 12.0 g/L due to better mixing by agitation at the higher cell density.     

Antioxidant property and production of exopolysaccharide from Armillaria mellea in submerged cultures: Effect of culture aeration rate

Effects of culture aeration rate on production and antioxidant property of exopolysaccharide (EPS) by Armillaria mellea were investigated in a 5‐L stirred‐tank bioreactor where an optimal biomass aeration rate of 1.2 vvm with 0.22 g/g cell yield and 0.6 vvm EPS formation rate with 7.66 mg/g product yield were achieved. A two‐stage aeration process to maximize the biomass and EPS productions proceeded with a 1.55‐fold enhancement (from 4.28 to 6.65 g/L) in biomass formation and a 2.68‐fold enhancement (from 86.9 to 233.2 mg/L) in the EPS production, as compared with those from the aeration rate of 0.3 vvm. The molecular weights of EPS in cultures of different aeration rates are closely correlated with their protein/polysaccharide ratios (R²=0.830) and EC₅₀ (EC₅₀, the effective concentration where the antioxidant property is 50%) values in antioxidant activity (R²=0.960), reducing power (R²=0.894) and chelating ability (R²=0.954). EPS from the two‐stage aeration rate culture shows a strong antioxidant property by the conjugated diene method, reducing power and chelating ability on ions. Therefore, we present results to regulate and to optimize A. mellea cultures to efficiently produce biomass and EPS. The fermented EPS has the potential to be used as for antioxidant‐related functional foods and pharmaceutical industries.     

Kinetic modeling and scale up of lipoic acid (LA) production from Saccharomyces cerevisiae in a stirred tank bioreactor

Scale up studies for production of lipoic acid (LA) from Saccharomyces cerevisiae have been reported in this paper for the first time. LA production in batch mode was carried out in a stirred tank bioreactor at varying agitation and aeration with maximum LA production of 512 mg/L obtained at 350 rpm and 25 % dissolved oxygen in batch culture conditions. Thus, LA production increased from 352 mg/L in shake flask to 512 mg/L in batch mode in a 5 L stirred tank bioreactor. Biomass production under these conditions was mathematically explained using logistic equation and data obtained for LA production and substrate utilization were successfully fitted using Luedeking–Piret and Mercier’s models. The kinetic studies showed LA production to be growth associated. Further enhancement of LA production was carried out using fed-batch (variable volume) and semi-continuous modes of fermentation. Semi-continuous fermentation with three feeding cycles of sucrose effectively increased the production of LA from 512 to 725 mg/L.     

Bromoperoxidase activity in microplantlet suspension cultures of the macrophytic red alga Ochtodes secundiramea.

Bromoperoxidase is an enzyme found in marine macroalgae that catalyzes the bromination of organic substrates. Photosynthetic microplantlet suspension cultures derived from the macrophytic red alga Ochtodes secundiramea were shown to possess bromoperoxidase. The optimum pH for O. secundiramea bromoperoxidase activity in cell-free extracts was 6.0, and the half-saturation constant for bromination of the exogeneous substrate monochlorodimedone (MCD) was 18 microM. O. secundiramea microplantlets were cultivated in a bubble-column photobioreactor at an incident light intensity of 38 microE x m(-2) x s(-1) (71% of light-saturated photosynthesis, 10:14 light:dark photoperiod), and the kinetics of cell growth and bromoperoxidase production were followed. At these conditions, the specific growth rate was 0.052 x day(-1). The lowest specific bromoperoxidase activity of 0.3 micromol MCD x g(-1) cell x min(-1) occurred during the midexponential phase of growth, and then increased steeply to 1.9 micromol MCD x g(-1) cell x min(-1) during the late stationary phase, suggesting that bromoperoxidase production was part of secondary metabolism. The estimated bromoperoxidase content in the cell mass at late stationary phase was 67 microg x g(-1) dry cell mass, demonstrating that bioreactor production of marine bromoperoxidase is feasible.     

Insights into large-scale cell-culture reactors: II. Gas-phase mixing and CO₂ stripping

Most discussions about stirred tank bioreactors for cell cultures focus on liquid-phase motions and neglect the importance of the gas phase for mixing, power input and especially CO(2) stripping. Particularly in large production reactors, CO(2) removal from the culture is known to be a major problem. Here, we show that stripping is mainly affected by the change of the gas composition during the movement of the gas phase through the bioreactor from the sparger system towards the headspace. A mathematical model for CO(2)-stripping and O(2)-mass transfer is presented taking gas-residence times into account. The gas phase is not moving through the reactor in form of a plug flow as often assumed. The model is validated by measurement data. Further measurement results are presented that show how the gas is partly recirculated by the impellers, thus increasing the gas-residence time. The gas-residence times can be measured easily with stimulus-response techniques. The results offer further insights on the gas-residence time distributions in stirred tank reactors.