Production of rosmarinic acid by<Emphasis Type="Italic">Lavandula vera</Emphasis> MM cell suspension in bioreactor: effect of dissolved oxygen concentration and agitation

The relationship between dissolved oxygen (DO) concentration, agitation rate and growth of Lavandula vera MM and rosmarinic acid biosynthesis was investigated in 3 l laboratory bioreactor. Lavandula vera MM cell suspension accumulated the highest amounts of biomass (34.8 g/l) and rosmarinic acid (1870.6 mg/l) on day 12 of cultivation at 50% dissolved oxygen and agitation speed 100 rpm and at 30% dissolved oxygen and agitation speed 300 rpm, respectively.     

Rosmarinic acid production by Lavandula vera MM cell suspension: the effect of temperature

Lavandula vera MM cell suspension, grown at 28 degrees C in a 3-l bioreactor, produced rosmarinic acid maximally at 3 g l(-1)) though most biomass (33.2 g dry wt l(-1)) was at 30 degrees C.     

Rosmarinic acid production by Lavandula vera MM cell-suspension culture

The time courses of growth and rosmarinic acid production by Lavandula vera MM cell suspension were investigated. The uptake of the main nutrients (sucrose, nitrogen, phosphorus, K, Ca, Mg) was followed during cultivation and the data on the physiology of the L. vera MM cell culture are presented. It was established that the cell culture synthesizes rosmarinic acid during the linear phase of growth for a relatively short period (between the 4th and 8th days of cultivation). The influence of sucrose concentration in the nutrient medium on cell growth and accumulation of rosmarinic acid by L. vera MM cell culture was investigated. The results showed that 7% sucrose in the nutrient medium ensured a steady growth of the cell suspension and increased the yield of rosmarinic acid (29.2 g/l dry biomass and 507.5 mg/l rosmarinic acid compared to 13.0 g/l dry biomass and 68.6 mg/l rosmarinic acid for the control cultivation with 3% sucrose). Received: 17 September 1996 / Received revision: 31 January 1997 / Accepted: 1 February 1997     

High density cultivation of Anchusa officinalis in a stirred-tank bioreactor with in situ filtration

Previously, Su et al. [Biotechnol Bioeng 42: 884–890 (1993)] reported improved production of rosmarinic acid by Anchusa officinalis in shake-flask cultures using a cultivation strategy that involved intermittent medium exchange. Implementation of this cultivation strategy in 2.5-1 stirred-tank bioreactor cultures is investigated in the present study. Intermittent cell/medium separation in the bioreactor was accomplished by means of automated in situ culture filtration. In the bioreactor culture, rosmarinic acid production was found very sensitive to agitation and aeration conditions as well as dissolved oxygen concentration. A maximum cell density of 35 g dry weight/l and a rosmarinic acid concentration of 3.7 g/l were obtained by maintaining the dissolved oxygen concentration above 30% air saturation, gradually raising the impeller tip speed from 34 cm/s to 72 cm/s, and keeping the aeration rate at 0.44 vvm while increasing the O₂: air ratio in the gas feed stream to 4:1. This result is comparable with the data obtained from shake-flask cultures using the same culture strategy.     

Multiple substrate growth kinetics of Leptothrix discophora SP-6

The growth parameters of Leptothrix discophora SP-6 were quantified on the basis of the steady-state concentrations and utilization rates of pyruvate, dissolved oxygen, and concentration of microorganisms in a chemostat operated at 25 degrees C, pH 7.2, and an agitation rate of 350 rpm. The results showed that the microbial growth was limited by both pyruvate and dissolved oxygen. A combined growth kinetics model using Monod growth kinetics for pyruvate and Tessier growth kinetics for oxygen showed the best correlation with the experimental data when analyzed using an interactive multiple substrate model. The growth kinetics parameters and the respective confidence limits, estimated using the Monte Carlo simulation, were mu(max) = 0.576 +/- 0.021 h(-1), K(sMp) = 38.81 +/- 4.24 mg L(-1), K(sTo) = 0.39 +/- 0.04 mg L(-1), Y(X/p) = 0.150 (mg microorganism mg(-1) pyruvate), Y(X/o) = 1.24 (mg microorganism mg(-1) oxygen), the maintenance factors for pyruvate and oxygen were m(p) = 0.129 (mg pyruvate consumed mg(-1) microorganism h(-1)) and m(o) = 0.076 (mg oxygen consumed mg(-1) microorganism h(-1)), respectively.     

Comparative study on the influence of dissolved oxygen control approaches on the microbial hyaluronic acid production of <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis>

Three different dissolved oxygen (DO) control approaches were proposed to improve hyaluronic acid (HA) production: a three-stage agitation speed control approach, a two-stage DO control approach, and an oxygen vector perfluorodecalin (PFC) applied approach. In the three-stage agitation speed control approach, agitation speed was 200 rpm during 0–8 h, 400 rpm during 8–12 h, and 600 rpm during 12–20 h. In the two-stage DO control strategy, DO was controlled at above 10% during 0–8 h and at 5% during 8–20 h. In the PFC applied approach, PFC (3% v/v) was added at 8 h. HA production reached 5.5 g/L in the three-stage agitation speed control culture model, and 6.3 g/L in two-stage DO control culture model, and 6.6 g/L in the PFC applied culture model. Compared with the other two DO control approaches, the PFC applied approach had a lower shear stress and thus a higher HA production was achieved.     

Effect of agitation and aeration on the citric acid production by Yarrowia lipolytica grown on glycerol

The effects of agitation rates from 400 to 900 rpm and aeration rates ranging from 0.18 to 0.6 vvm on biomass and citric acid production on glycerol media by acetate-negative mutants of Yarrowia lipolytica, Wratislavia 1.31 and Wratislavia AWG7, in batch culture were studied. The agitation rates of 800 and 900 rpm (at a constant aeration rate of 0.36 vvm) and aeration rates within the range of 0.24-0.48 vvm (at a constant agitation rate of 800 rpm), which generated dissolved oxygen concentration (DO) higher than 40%, were found the best for citric acid biosynthesis from glycerol. An increase in agitation rate (higher than 800 rpm) and aeration rate (higher than 0.36 vvm) had no impact on DO and citric acid production. The highest citric acid concentration (92.8 g/L) and yield (0.63 g/g) were obtained with Wratislavia 1.31 strain at 0.24 vvm. The highest volumetric citric acid production rate (1.15 g/Lh) and specific citric acid production rate (0.071 g/gh) were reached at 0.48 vvm.     

Influence of oxygen transfer on <Emphasis Type="Italic">Pseudomonas putida</Emphasis> effects on growth rate and biodesulfurization capacity

The growth rate and desulfurization capacity accumulated by the cells during the growth of Pseudomonas putida KTH2 under different oxygen transfer conditions in a stirred and sparged tank bioreactor have been studied. Hydrodynamic conditions were changed using different agitation conditions. During the culture, several magnitudes associated to growth, such as the specific growth rate, the dissolved oxygen concentration and the carbon source consumption have been measured. Experimental results indicate that cultures are influenced by the fluid dynamic conditions into the bioreactor. An increase in the stirrer speed from 400 to 700 rpm has a positive influence on the cell growth rate. Nevertheless, the increase of agitation from 700 to 2000 rpm hardly has any influence on the growth rate. The effect of fluid dynamics on the cells development of the biodesulfurization (BDS) capacity of the cells during growth is different. The activities of the intracellular enzymes involved in the 4S pathway change with dissolved oxygen concentration. The enzyme activities have been evaluated in cells at several growth time and different hydrodynamic conditions. An increase of the agitation from 100 to 300 rpm has a positive influence on the development of the overall BDS capacity of the cells during growth. This capacity shows a decrease for higher stirrer speeds and the activity of the enzymes monooxygenases DszC and DszA decreases dramatically. The highest value of the activity of DszB enzyme was obtained with cells cultured at 100 rpm, while this activity decreases when the stirrer speed was increased higher than this value.     

Enhancement of pyruvate production by Torulopsis glabrata using a two-stage oxygen supply control strategy

The effect of agitation speeds on the performance of producing pyruvate by a multi-vitamin auxotrophic yeast, Torulopsis glabrata, was investigated in batch fermentation. High pyruvate yield on glucose (0.797 g g(-1)) was achieved under high agitation speed (700 rpm), but the glucose consumption rate was rather low (1.14 g l(-1) h(-1)). Glucose consumption was enhanced under low agitation speed (500 rpm), but the pyruvate yield on glucose decreased to 0.483 g g(-1). Glycerol production was observed under low agitation speed and decreased with increasing agitation speed. Based on process analysis and carbon flux distribution calculation, a two-stage oxygen supply control strategy was proposed, in which the agitation speed was controlled at 700 rpm in the first 16 h and then switched to 500 rpm. This was experimentally proven to be successful. Relatively high concentration of pyruvate (69.4 g l(-1)), high pyruvate yield on glucose (0.636 g g(-1)), and high glucose consumption rate (1.95 g l(-1)h(-1)) were achieved by applying this strategy. The productivity (1.24 g l(-1) h(-1)) was improved by 36%, 23% and 31%, respectively, compared with fermentations in which agitation speeds were kept constant at 700 rpm, 600 rpm, and 500 rpm. Experimental results indicate that the difference between the performances for producing pyruvate under a favorable state of oxygen supply (dissolved oxygen concentration >50%) was caused by the different regeneration pathways of NADH generated from glycolysis.     

Factors influencing the production of a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid, by Pseudomonas aeruginosa PR3 (NRRL B-18602) in batch cultures

Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor. Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28 degrees C and 300 rpm for 16-20 h and the culture was adjusted to pH 7 prior to substrate addition. Increases of medium volume and substrate concentration failed to enhance yield. When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable. This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture. Under the optimal conditions of 650 rpm, 28 degrees C, and 40-60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel. This is the first report on a successful scale-up production of DOD.     

Radical quenching by rosmarinic acid from Lavandula vera MM cell culture

This study was conducted to evaluate the radical scavenging capacities of extracts and preparations from a Lavandula vera MM plant cell culture with different rosmarinic acid content and to compare them with pure rosmarinic and caffeic acids as well. The methods, which were used are superoxide anion and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radicals scavenging assays. Results showed that extracts and preparations from Lavandula vera MM possess strong radical scavengers, as the best both radical scavengers appeared to be the fractions with enriched rosmarinic acid content, obtained after ethylacetate fractioning (47.7% inhibition of superoxide radicals and 14.2 microM 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid equivalents, respectively). These data reveal the possibilities for application of these preparations as antioxidants.     

Effects of methocel A15LV, polyethylene glycol, and polyvinyl alcohol on CD13 and CD33 receptor surface content and metabolism of HL60 cells cultured in stirred tank bioreactors

Flow cytometry was used to examine the effect of hydrodynamic forces in a stirred tank bioreactor on the CD13 and CD33 receptor surface content of HL60 (human promyelocytic leukemia) cells. A step increase in agitation rate from 80 to 400 rpm reduced the HL60 cell apparent growth rate and increased the CD13 receptor surface content per cell, on average, by 95%. In contrast, this step increase in agitation rate to 400 rpm decreased the CD33 receptor surface content per cell, on average, by 10%. The protective effects of 0.1% Methocel A15LV, polyethylene glycol (PEG), and polyvinyl alcohol (PVA) on CD13 and CD33 receptor surface content were examined under agitation at 300 rpm in parallel 2 L bioreactor runs. The average CD33 receptor surface content was unaffected by the presence of Methocel A15LV or PEG, while PVA had a slight protective effect. In contrast, in terms of CD13 receptor content, HL60 cells agitated at 300 rpm with Methocel A15LV, PEG, or PVA behaved like cells agitated at 80 rpm with no media additives (McDowell and Papoutsakis, 1998). That is, Methocel A15LV, PEG, and PVA prevented the transduction of mechanical forces which affect CD13 cell content. HL60 cells cultured with 0.1% A15LV, PEG or PVA under conditions of mild agitation (60 rpm) in spinner flasks exhibited glucose consumption and lactate production rates that were approximately 20% lower than values of cultures containing no additive. Under conditions of agitation at 300 rpm in the 2 L bioreactor, the presence of A15LV, PEG, and PVA reduced the HL60 glucose consumption and lactate production rates by approximately 50%. Thus, media additives can dramatically reduce lactate accumulation in agitated bioreactors due to cell growth, in addition to providing protection from cellular injury.     

Effect of aeration on denitrification byOchrobactrum anthropi SY509

Aeration was found to affect the biological denitrification byOchrobactrum anthropi SY509. Although cell growth was vigorous under 1 vvm of aeration and an agitation speed of 400 rpm in a 3-L jar fermentor, almost no nitrate was removed. Yet under low agitation speeds (100, 200, and 300 rpm), denitrification occurred when the dissolved oxygen was exhausted shortly after the inoculation of the microorganism.Ochrobactrum anthropi SY509 was found to express highly active denitrifying enzymes under anaerobic conditions. The microorganism also synthesized denitrifying enzymes under aerobic conditions (1 vvm and 400 rpm), yet their activity was only 60% of the maximum level under anaerobic conditions and the nitrate removal efficiency was merely 15%. However, although the activities of the denitrifying enzymes were inhibited in the presence of oxygen, they were fully recovered when the conditions were switched to anaerobic conditions.     

Twenty-four-well plate miniature bioreactor high-throughput system: assessment for microbial cultivations

High-throughput (HT) miniature bioreactor (MBR) systems are becoming increasingly important to rapidly perform clonal selection, strain improvement screening, and culture media and process optimization. This study documents the initial assessment of a 24-well plate MBR system, Micro (micro)-24, for Saccharomyces cerevisiae, Escherichia coli, and Pichia pastoris cultivations. MBR batch cultivations for S. cerevisiae demonstrated comparable growth to a 20-L stirred tank bioreactor fermentation by off-line metabolite and biomass analyses. High inter-well reproducibility was observed for process parameters such as on-line temperature, pH and dissolved oxygen. E. coli and P. pastoris strains were also tested in this MBR system under conditions of rapidly increasing oxygen uptake rates (OUR) and at high cell densities, thus requiring the utilization of gas blending for dissolved oxygen and pH control. The E. coli batch fermentations challenged the dissolved oxygen and pH control loop as demonstrated by process excursions below the control set-point during the exponential growth phase on dextrose. For P. pastoris fermentations, the micro-24 was capable of controlling dissolved oxygen, pH, and temperature under batch and fed-batch conditions with subsequent substrate shot feeds and supported biomass levels of 278 g/L wet cell weight (wcw). The average oxygen mass transfer coefficient per non-sparged well were measured at 32.6 +/- 2.4, 46.5 +/- 4.6, 51.6 +/- 3.7, and 56.1 +/- 1.6 h(-1) at the operating conditions of 500, 600, 700, and 800 rpm shaking speed, respectively. The mixing times measured for the agitation settings 500 and 800 rpm were below 5 and 1 s, respectively.     

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.     

Development of a helical-ribbon impeller bioreactor for high-density plant cell suspension culture

A double helical-ribbon impeller (HRI) bioreactor with a 11-L working volume was developed to grow high-density Catharanthus roseus cell suspensions. The rheological behavior of this suspension was found to be shear-thinning for concentrations higher than 12 to 15 g DW . L(-1). A granulated agar suspension of similar rheological properties was used as a model fluid for these suspensions. Mixing studies revealed that surface baffling and bottom profiling of the bioreactor and impeller speeds of 60 to 150 rpm ensured uniform mixing of suspensions. The HRI power requirement was found to increase singnificantly for agar suspensions higher than 13 g DW . L(-1), in conjunction with the effective viscosity increase. Oxygen transfer studies showed high apparent surface oxygen transfer coefficients (k(L)a approximately 4 to 45 h(-1)) from agar suspensions of 30 g DW . L(-1) to water and for mixing speeds ranging from 120 to 150 rpm. These high surface k(I)a values were ascribed to the flow pattern of this bioreactor configuration combined with surface bubble generation and entrainment in the liquid phase caused by the presence of the surface baffles. High-density C. roseus cell suspension cultures were successfully grown in this bioreactor without gas sparging. Up to 70% oxygen enrichment of the head space was required to ensure sufficient oxygen supply to the cultures so that dissolved oxygen concentration would remain above the critical level (>/=10% air saturation). The best mixing speed was 120 rpm. These cultures grew at the same rate ( approximately 0.4 d(-1)) and attained the same high biomass concentrations ( approximately 25 to 27 g DW . L(-1), 450 to 500 g filtered wet biomass . L(-1), and 92% to 100% settled wet biomass volume) as shake flask cultures. The scale-up potential of this bioreactor configuration is discussed.     

Production of anti‐inflammatory compounds in Sphaeralcea angustifolia cell suspension cultivated in stirred tank bioreactor

Sphaeralcea angustifolia is a plant used for the treatment of inflammatory processes. Scopoletin, tomentin, and sphaeralcic acid were identified as the compounds with anti‐inflammatory and immunomodulatory effects. Successful establishment of the cell culture in Erlenmeyer flasks has been reported previously. The aim of this study was to evaluate the ability of cells in suspension from S. angustifolia grown in a stirred tank bioreactor and demonstrate their capacity to produce bioactive compounds. Cells in suspension grown at 200 rpm reached a maximal cell biomass in dry weight at 19.11 g/L and produced 3.47 mg/g of sphaeralcic acid. The mixture of scopoletin and tomentin was only detected at the beginning of the culture (12.13 μg/g). Considering that the profile of dissolved oxygen during the cultures was lesser than 15%, it is possible that the low growth at 100 rpm could be due to oxygen limitations or to cell sedimentation. At 400 rpm, a negative effect on cell viability could be caused by the increase in the hydrodynamic stress, including the impeller tip, average shear rate, and Reynolds number. The sphaeralcic acid content in the cell suspension of S. angustifolia obtained in the bioreactor was two orders of magnitude greater than that reported for the culture grown in Erlenmeyer flasks.     

A novel centrifugal impeller bioreactor. II. Oxygen transfer and power consumption

The effects of the impeller configuration, aeration rate, and agitation speed on oxygen transfer coefficient K(L)a were studied in a newly designed centrifugal impeller bioreactor (5-L). The oxygen transfer rates in the novel bioreactor were also compared with those in a cell-lift bioreactor with comparable dimensions. The cell-lift impeller produced much higher surface oxygen transfer rates than the centrifugal one at an agitation speed over 200 rpm. This result was in good agreement with our observation that the cell-lift impeller produced much higher unfavorable turbulence. In addition, the experiments using granulated agar particles as pseudo plant cells indicated that the K(L)a value decreased steadily with an increase in agar particle concentration, and the centrifugal impeller still demonstrated a larger K(L)a than the cell lift up to a high pseudo cell concentration of 19.5 g dry weight (DW)/L (under 150 rpm and 0.20 vvm) or 22.3 g DW/L (under 200 rpm and 0.20 vvm). Furthermore, the correlation between power number and impeller Reynolds number for both the centrifugal and the cell-lift impellers was successfully obtained, which could be used for predicting the power input required by each impeller. From the results obtained, the centrifugal impeller bioreactor is expected to have great potential in its application to shear-sensitive biological systems.     

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.