K L a Evaluation during the course of fermentation using data reconciliation techniques

The oxygen mass transfer coefficient often serves to compare the efficiency of bioreactors and their mixing devices as well as being an important scale-up factor. In submerged fermentation, four methods are available to estimate the overall oxygen mass transfer coefficient (K_La): the dynamic method, the stationary method based on a previous determination of the oxygen uptake rate (Q_O2X), the gaseous oxygen balance and the carbon dioxide balance. Each method provides a distinct estimation of the value of K_La. Data reconciliation was used to obtain a more probable value of K_La during the production of Saccharomyces cerevisiae, performed in 22.5-l fed-batch bioreactor. The estimate of K_La is obtained by minimising an objective function that includes measurement terms and oxygen conservation models, each being weighted according to their level of confidence. Weighting factors of measurement terms were taken as their respective inverse variance whereas weighting factors of oxygen conservation models were obtained using Monte Carlo simulations. Results show that more coherent and precise estimations of K_La are obtained.     

Laboratory scale equipment for the determination of k L a in bio-reactors

The study of the oxygen transfer rate (O.T.R) in reactors designed for cell cultivation and enzyme reaction is a difficult task. In this work a bio-reactor for acetic acid fermentation purposes is studied by using the static gassing out method for K_La evaluation. Results obtained prove that K_La shows linear dependence versus operation temperature.     

From field sampling to pneumatic bioreactor mycelia production of the ectomycorrhizal mushroom Laccaria trichodermophora

In order to increase survival rates of greenhouse seedlings destined for restoration and conservation programs, successful mycorrhization of the seedlings is necessary. To reforest forest ecosystems, host trees must be inoculated with ectomycorrhizal fungi and, in order to guarantee a sufficient supply of ectomycorrhizal inoculum, it is necessary to develop technologies for the mass production of ectomycorrhizal fungi mycelia. We selected the ectomycorrhizal fungus Laccaria trichodermophora, due to its ecological traits and feasible mycelia production in asymbiotic conditions. Here, we report the field sampling of genetic resources, as well as the highly productive nutritional media and cultivation parameters in solid cultures. Furthermore, in order to achieve high mycelial production, we used strain screening and evaluated pH, carbon source concentration, and culture conditions of submerged cultures in normal and baffled shake flasks. The higher productivity culture conditions in shake flasks were selected for evaluation in a pneumatic bioreactor, using modified BAF media with a 10 g/L glucose, pH 5.5, 25 °C, and a volumetric oxygen transfer coefficient (K_La) of 36 h⁻¹. Under those conditions less biomass (12–37 %) was produced in the pneumatic bioreactor compared with the baffled shake flasks. This approach shows that L. trichodermophora can generate a large biomass concentration and constitute the biotechnological foundation of its mycelia mass production.     

Statistical media optimization and production of ITS alpha-amylase from Aspergillus oryzae in a bioreactor

The production of an intermediate temperature-stable (ITS) α-amylase from Aspergillus oryzae was studied by using a central composite design with three independent variables, viz., starch, yeast extract, and K₂HPO₄. The model equation provided a suitable model for the response surface for α-amylase production, and, from the optimal concentrations of the medium components, a model was predicted, which was then used for enzyme production in a 150-L bioreactor. In the bioreactor studies, the enzyme yields (161 U/ml) were similar to that of the shake flask (133 U/ml); however, the time required for maximum α-amylase production in the bioreactor was reduced to 48 h compared with 120 h in shake flask cultures. An increased level of phosphate in the medium and low inoculum size were necessary to control the excessive foaming in the bioreactor; however, control of the pO₂ level and agitation was not mandatory for enzyme production. The peak enzyme production coincided with the increase in pH of the fermentation broth and was maximal when the pH of the system was above 7.5. Thus, in the present study, pH acted as an indicator of the initiation or end of the enzyme synthesis or of the fermentation cycle. Received: 20 November 2001 / Accepted 31 December 2001     

Feasibility of culturing C2C12 mouse myoblasts on glass microcarriers in a continuous stirred tank bioreactor

Commercial culturing of mammalian cell lines is increasing in importance as more biological products unique to mammals are being produced in genetically altered mammalian cells. Most mammalian cells are anchorage dependent, so they must be cultured on a support matrix. This limitation, along with the requirement of a low shear environment, severely effects the scale-up of bench-scale culture systems. The need to culture mammalian cells on a support matrix limits the increase in cell population to a factor of 10-20 before growth virtually stops due to contact inhibition. Commercial culturing systems for anchorage dependent cells are batch processes because of the combination of contact inhibition and support matrix requirements. Development of a continuous bioreactor system could allow both unlimited scale-up and continuous cell-mass production. To design a continuous reactor, a mathematical model to predict the reactor performance should be developed. This paper addresses the development of a mathematical model for predicting continuous bioreactor performance. It was found that anchorage dependent C2C12 mouse myoblast cells, a continuous cell line, followed Monod kinetics for glucose consumption and cell mass production in batch flask experiments, with w_max = 0.040 hr^у and K_m = 2.5 mM. Furthermore, it was found that these parameters could be used to predict the glucose consumption in a continuous bioreactor operated with constant feed of seeded microcarriers operated at two different residence times. The success of this model implies the possibility of developing a continuous cell harvesting and reinoculation system using a microcarrier bioreactor to produce cell mass.     

Limitation of stomatal conductance by hydraulic traits: sensing or preventing xylem cavitation?

We tested the hypothesis that hydraulic conductance per unit leaf surface area of plant shoots (K_SL) determines the maximum diurnal stomatal conductance (g_L) that can be reached by plants growing in the field. A second hypothesis was tested that some xylem cavitation cannot be avoided by transpiring plants and might act as a signal for regulating g_L. Eleven woody species were studied, differing from each other with respect to taxonomy, wood anatomy and leaf habit. Maximum diurnal g_L, transpiration rate (E_L), pre-dawn and minimum diurnal leaf water potential (O_pd and O_min, respectively) were measured in the field. The critical O level at which stem cavitation was triggered (O_cav) was measured on detached branches, using the acoustic method. A high-pressure flow meter was used to measure maximum K_SL of 1-year-old shoots. Both g_L and E_L were positively related to K_SL. The whole-plant hydraulic conductance per unit leaf area (K_WL) of all the species studied, calculated as the ratio of E_L to (O (=O_pd-O_min) was closely related to K_SL. In every case, O_min (ranging between -0.85 and -1.35 MPa in the different species) dropped to the O_cav range or was <O_cav (ranging between -0.71 and -1.23 MPa), thus suggesting that some cavitation-induced embolism could not be avoided. The possibility is discussed that some cavitation-induced reduction in K_SL is the signal for stomatal closure preventing runaway embolism. The lack of correlation of g_L to O_cav is discussed in terms of the inconsistency of O_cav as an indicator of the vulnerability of plants to cavitation. No differences in hydraulic traits were observed between evergreen and deciduous species.     

Modeling of growth and laccase production by Pycnoporus sanguineus

Production of extracellular laccase by the white-rot fungus Pycnoporus sanguineus was examined in batch submerged cultures in shake flasks, baffled shake flasks and a stirred tank bioreactor. The biomass growth in the various culture systems closely followed a logistic growth model. The production of laccase followed a Luedeking-Piret model. A modified Luedeking-Piret model incorporating logistic growth effectively described the consumption of glucose. Biomass productivity, enzyme productivity and substrate consumption were enhanced in baffled shake flasks relative to the cases for the conventional shake flasks. This was associated with improved oxygen transfer in the presence of the baffles. The best results were obtained in the stirred tank bioreactor. At 28 °C, pH 4.5, an agitation speed of 600 rpm and a dissolved oxygen concentration of ~25 % of air saturation, the laccase productivity in the bioreactor exceeded 19 U L^?1 days^?1, or 1.5-fold better than the best case for the baffled shake flask. The final concentration of the enzyme was about 325 U L^?1.     

Oxygen transfer characteristics of a centrifugal, packed-bed reactor during viscous xanthan fermentation

The oxygen transfer properties of a novel, centrifugal, packed-bed reactor (CPBR) during viscous xanthan fermentation were determined with respect to the effects of the arrangement of the centrifugal, packed bed (CPB) and the recirculation loop (RL). Characterized by the maximum volumetric transfer coefficient (k_La) in xanthan broth, the aeration efficiency of CPBR was compared to those in stirred-tank reactors (STR) equipped with disc turbines (DT) or marine propellers (MP), and to that in a water-in-oil emulsion (WIO). As expected, STR-WIO showed the highest k_La (0.038 s^-1 at 2%) among all systems studied due to reduced broth viscosity; however, practical difficulties exist in product recovery. It was found that, at 3.5% xanthan the k_La in CPBR (0.018 s^-1) was higher than that of STR (0.005 s^-1) and close to that of STR-WIO (0.020 s^-1), indicating improved oxygen transfer at such a xanthan concentration. The exterior baffles along the rotating fibrous matrix offer additional agitation in the viscous broth. A gas-continuous arrangement, in which the CPB was kept above the broth, was able to elevate k_La to 0.023 s^-1, higher than that of STR-WIO. The external RL operated by a peristaltic pump was found to play an important role in CPBR aeration by providing better gas-liquid contact. With the improved oxygen transfer efficiency in CPBR at high xanthan concentrations, the CPBR system is practically the preferred system for xanthan fermentation. The characteristic roles of CPB arrangement and the RL should be considered primarily during scale-up operation.     

Anthraquinones production in Rubia tinctorum cell suspension cultures: Down scale of shear effects

The effect of turbulence on suspended cells is one of the most complex problems in the scale-up of cell cultures. In the present paper, a direct comparison of the effects of turbulence on suspension cultures of Rubia tinctorum in a standard bioreactor and in shake flask cultures was done. A procedure derived from the well known global method proposed by Nishikawa et al. (1977) [39] was applied. Standard flasks and four-baffled shake flasks were used. The effect of turbulence and light irradiation on cell viability, biomass, and anthraquinones (AQs) production was evaluated. The biomass concentration and AQs production obtained using baffled shake flasks agitated at 360 rpm were similar to that achieved in R. tinctorum suspension cultures growing in a stirred tank bioreactor operating at 450 rpm, previously published (Busto et al., 2008 [17]). The effect of light on AQs production was found to be very significant, and a difference of up to 48% was found in cells with and without illumination after 7 days of culture. It is concluded that this down-scaled and simple flask culture system is a suitable and valid small scale instrument for the study of intracellular mechanisms of turbulence-induced AQs production in R. tinctorum suspension cultures.     

Kinetics of cell growth and cyclosporin A production by Tolypocladium inflatum when scaling up from shake flask to bioreactor

The kinetics of cell growth and Cyclosporin A (Cyc A) production by Tolypocladium inflatum were studied in shake flasks and bioreactors under controlled and uncontrolled pH conditions. In the case of the shake flask, the production time was extended to 226 h and the maximal antibiotic concentration was 76 mg/l. When scaling up the cultivation process to a bioreactor level, the production time was reduced to only 70 h with a significant increase in both the cell growth and the antibiotic production. The maximal dry cell weights in the case of the controlled pH and uncontrolled pH cultures in the bioreactor were 22.4 g/l and 14.2 g/l, respectively. The corresponding maximal dry cell weight values did not exceed 7.25 g/l with the shake flask cultures. The maximal values for Cyc A production were 144.72 and 131.4 mg/l for the controlled and uncontrolled pH cultures, respectively. It is also worth noting that a significant reduction was observed in both the dry cell mass and the antibiotic concentration after the Cyc A production phase, whereas the highest rate of antibiotic degradation was observed in the stirred tank bioreactor with an uncontrolled pH. Morphological characterization of the micromorphological cell growth (mycelial/pellet forms) was also performed during cultivation in the bioreactor.     

A novel parallel shaken bioreactor system for continuous operation

A novel continuous bioreactor system was developed as a shaken culture vessel for the investigation of the growth kinetics and product formation of microorganisms in milliscale. The novel bioreactor system mainly consists of a specially designed 250-mL shake flask with two inlets, one for gas supply and one for medium supply, and one combined outlet on the side of flask for exhaust gas and culture liquid. As a result of the circulating motion of the fermentation broth in the shake flask, the maximum liquid height reaches the edge of the outlet and the fermentation broth is accelerated into the outlet by centrifugal force. Additionally, the excess fermentation broth leaving the culture vessel is continuously driven by the exhaust gas. Because of the small scale and the simple handling it is possible to operate many of these shaken bioreactor vessels simultaneously. By using parallel vessels operated at different dilution rates on the same shaker, the data for a complete biomass over dilution rate (X-D) diagram of a biological culture can be evaluated in an efficient manner, thus saving money, materials, and time. Continuous fermentations of the yeast Saccharomyces cerevisiae H1022 (ATCC 32167) in the shaken bioreactor system and in a conventional stirred tank fermentor showed very similar results.     

Process conditions affecting proteolysis of recombinant proteins in Escherichia coli

The effects of process conditions on proteolysis of three recombinant IgG-binding proteins ZT3, ZZT3 and protein A in E. coli were studied. SDS/PAGE shows that the relative amount of degradation intermediates of ZT3 in a shake flask cultivation is much higher than that in a bioreactor culture. The rate of proteolysis of ZZT3 and protein A was also higher in shake flask cultures than in bioreactor cultures. The proteolysis rate constant of ZZT3 was 12/h in a shake flask but only 2.1/h in a bioreactor. Corresponding values for protein A were 2.4/h and 1.0/h, respectively. High proteolysis rate constants correlated with lower product yields.     

Effect of gaseous composition on cell growth and secondary metabolite production in suspension culture of Stizolobium hassjoo cells

The gaseous composition is an important factor affecting the performance of plant cell cultures. Gaseous metabolites, especially O₂, CO₂ and C₂H₄, play important roles in cell physiology. Forced aeration in bioreactors usually results in poor cell growth and secondary metabolite production. In this work, the effects of gaseous metabolites on cell growth, secondary metabolite formation as well as PPO activity were investigated with respect to Stizolobium hassjoo cell culture producing l-DOPA (3,4-dihydroxyphenylalanine). A device allowing the control of the partial pressures of gaseous metabolites in shake flasks was designed. In addition, a recirculating gas system with a P_O2 controller was designed for a bioreactor. This device could maintain constant P_O2 and P_CO2 in the bioreactor headspace. The results showed that the highest l-DOPA content was attained at P_O2=0.30 atm. Higher P_O2 values retarded cell growth and increased the pH of the culture broth. High P_O2 also enhanced the formation of ethylene and inhibited l-DOPA formation. Carbon dioxide concentrations lower than 5% enhanced cell growth and l-DOPA formation. Cell growth was retarded by 0.3 ppm of ethylene in 2~5 carbon dioxide. Oxygen concentration and D.O. in the broth could be controlled at constant levels in the recirculating culture system. Enrichment of P_O2 up to 0.3 atm during the later stage of cultivation facilitated l-DOPA formation. The interaction among the gaseous metabolites and their influences on cell metabolism and l-DOPA formation were elucidated. This information will facilitate the rational operation of plant cell culture systems producing secondary metabolites.     

Production of anthraquinones by immobilized Frangula alnus Mill. plant cells in a four-phase air-lift bioreactor

 The production of anthraquinones by Frangula alnus Mill. plant cells was used as a model system to evaluate the performance of a liquid-liquid extractive product-recovery process. The shake flask experiments have shown higher production of anthraquinones in cell suspension and flask cultures of calcium-alginate-immobilized cells when silicone oil was incorporated into the medium, compared to a control without silicone oil. An external-loop air-lift bioreactor, developed and designed for the production and simultaneous extraction of extracellular plant cell products, was regarded as a four-phase system, with dispersed gas, non-aqueous solvent and calcium-alginate-immobilized plant cells in Murashige and Skoog medium. Continuous extraction of anthraquinones by silicone oil and n-hexadecane inside the bioreactor resulted in 10–30 times higher cell productivity, compared to that of immobilized cells in a flask. Based on the mixing pattern, immobilized biocatalyst extraparticle and intraparticle diffusional constraints and the kinetics of growth, substrate consumption and product formation, a mathematical model was developed to describe the time course of a batch plant cell culture. The model showed satisfactory agreement with four sets of shake flask experiments and three bioreactor production cycles. Received: 18 March 1994/Received revision: 20 September 1994/Accepted: 28 September 1994     

Tight control of growth and cell differentiation in photoautotrophically growing moss (<Emphasis Type="Italic">Physcomitrella</Emphasis><Emphasis Type="Italic">patens</Emphasis>) bioreactor cultures

The use of the moss Physcomitrella patens as a production system for heterologous proteins requires highly standardised culture conditions. For this purpose a semi-continuous photoautotrophic bioreactor culture of Physcomitrella was established. This culture grew stably for 7 weeks in a 5-l bioreactor with a dilution rate of 0.22/day. Enrichment of the air for aeration in a batch bioreactor culture with 2% (v/v) CO₂ resulted in an increase in the specific growth rate to 0.57/day. Changes in the pH of the semi-continuous bioreactor culture medium between pH 4.5 and pH 7.0 influenced protonema differentiation; however it did not negatively affect the growth rate compared to uncontrolled pH. The advantages of Physcomitrella as a system for the production of heterologous proteins in plants are discussed.     

Scale up studies for the production of biosurfactant in packed column bioreactor

Biosurfactants capable of emulsifying pesticides have great potential to assist in microbial degradation of the pesticides. Solid State Fermentation (SSF) due to several advantages, is one of the efficient ways of producing these surfactants and seldom receives attention for commercial exploitation. In this study, a packed column bioreactor with wheat bran as the raw material and Bacillus subtilis has been used to produce a biosurfactant specific to disperse Fenthion, an organophosphrous pesticide. The emulsifier activity (EA) and surface tension from the packed column bioreactor were compared with flask fermentation experiments, which served as control. Airflow rate in the packed column bioreactor was varied from 10-20 l/min. Maximum EA and minimum surface tension occurred at airflow rate of 20 l/min. Peak EA in the control was 1.2 at 29 h while it was 1.9 in the bioreactor. The least surface tension of 24 dynes/cm was noticed at 54 h in the bioreactor, which was 33% better than the control at the same time period. The results indicate that the packed column bioreactor can become a more acceptable solid state fermentation system for commercial exploitation of Fenthion specific biosurfactant production.     

Effect of the impeller-sparger configuration over Trichoderma harzianum growth in four-phases cultures under constant dissolved oxygen

Three impeller-sparger configurations were used to evaluate the effect of different hydrodynamic conditions over fungal growth in rheologically complex cultures of Trichoderma harzianum using castor oil as sole carbon source. Three spargers (ring, sintered and 5-orifice) in combination with a turbine impeller system "TIS" (two Rushton turbines) or a hybrid impeller system "HIS" (Rushton turbine and a marine propeller as lower and upper impellers) were used. Their performance was assessed in terms of the response towards disturbance (PID oxygen control settings) and oxygen mass transfer (k_La). To avoid oxygen limitations, all cultures were controlled at 10% DOT by gas blending. Top to bottom mixing, and hence bulk blending, was improved when the - axial flow - HIS was used, ensuring phase interaction and substrate (oil) circulation. The 5-orifice sparger in combination with the TIS configuration yielded the longest lag phase and lowest k_La due to poor bulk blending and to the low gas-liquid interfacial area developed. The highest k_La was achieved with the sintered sparger-HIS probably due to considerable interfacial bubble area enhancement. However, growth limitation occurred as consequence of poor substrate availability as a stable air-oil emulsion was formed at the top of the tank. The best compromise between bulk blending (phase interaction), oxygen transfer (k_La) and fungal growth (growth rate) was achieved with the ring sparger-HIS configuration.     

Detection of ginkgolide A in Ginkgo biloba cell cultures

Ginkgo biloba cells were cultured in two 500 mL shake flasks and in 2 L and 6 L immobilization bioreactors using MS medium supplemented with 1 mg.L^–1 NAA, 0.1 mg.L^–1 K and 30 g.L^–1 sucrose. Specific growth rates were 0.06 d^–1, 0.11 d^–1 and 0.07 d^–1 for the 2 L and 6 L bioreactors and shake flask cultures, respectively. Extracellular phosphate, nitrate, ammonium and carbohydrate uptake rates of the bio reactor cultures were approximately 17 to 39% slower than those of shake flask cultures. The specific oxygen uptake and carbon dioxide transfer rates of immobilized Ginkgo biloba cells ranged from 0.027 to 0.041 mmol O₂.g^–1.d.w.hr^–1 (maximum uptake at 14 days) and 0.020 to 0.057 mmol CO₂g. ^–1.d.w.hr^–1 (maximum production at 14 days). Extracts from the biomass of the two immobilized and shake flask suspension cultures were analysed for ginkgolide A by GC-MS. Yields of 7, 17, 19 and 7 ng.g. ^–1d.w. of ginkgolide A were determined for shake flask 1, shake flask 2 and the 2 L and 6 L immobilized cultures, respectively. Traces of ginkgolide B were detected with the signal to noise ratio, however, being too low for positive confirmation of this last product.Abbreviations CTR Carbon dioxide transfer rate - DO Dissolved oxygen - g.d.w. Gram dry weight - GA Ginkgolide A - GB Ginkgolide B - GC Gas chromatography - GC-MS Gas chromatography-mass spectrometry - HPLC High performance liquid chromatography - K Kinetin - MS Murashige and Skoog salt medium - N₁K₁MS Complete Murashige and Skoog medium supplemented with 1 mg.L^–1 NAA, 0.1 mg.L^–1 K and 30g.L^–1 sucrose - NAA Naphthaleneacetic acid - OTR Oxygen transfer rate - PAF Platelet Aggregating Factor - q_CO2 Specific carbon dioxide production rate - q_O2 Specific oxygen uptake rate - u Specific growth rate     

Influence of climate-driven shifts in biomass allocation on water transport and storage in ponderosa pine

Conifers decrease the amount of biomass apportioned to leaves relative to sapwood in response to increasing atmospheric evaporative demand. We determined how these climate-driven shifts in allocation affect the aboveground water relations of ponderosa pine growing in contrasting arid (desert) and humid (montane) climates. To support higher transpiration rates, a low leaf:sapwood area ratio (A_L/A_S) in desert versus montane trees could increase leaf-specific hydraulic conductance (K_L). Alternatively, a high sapwood volume:leaf area ratio in the desert environment may increase the contribution of stored water to transpiration. Transpiration and hydraulic conductance were determined by measuring sap flow (J_S) and shoot water potential during the summer (June-July) and fall (August-September). The daily contribution of stored water to transpiration was determined using the lag between the beginning of transpiration from the crown at sunrise and J_S. In the summer, mean maximum J_S was 31.80LJ.74 and 24.34Dž.05 g m^-2 s^-1 for desert and montane trees (a 30.6% difference), respectively. In the fall, J_S was 25.33NJ.52 and 16.36dž.64 g m^-2 s^-1 in desert and montane trees (a 54.8% difference), respectively. J_S was significantly higher in desert relative to montane trees during summer and fall (P<0.05). Predawn and midday shoot water potential and sapwood relative water content did not differ between environments. Desert trees had a 129% higher K_L than montane trees in the summer (2.41᎒^-5 versus 1.05᎒^-5 kg m^-2 s^-1 MPa^-1, P<0.001) and a 162% higher K_L in the fall (1.97᎒^-5 versus 0.75᎒^-5 kg m^-2 s^-1 MPa^-1, P<0.001). Canopy conductance decreased with D in all trees at all measurement periods (P<0.05). Maximum g_C was 3.91 times higher in desert relative to montane trees averaged over the summer and fall. Water storage capacity accounted for 11 kg (11%) and 10.6 kg (17%) of daily transpiration in the summer and fall, respectively, and did not differ between desert and montane trees. By preventing xylem tensions from reaching levels that cause xylem cavitation, high K_L in desert ponderosa pine may facilitate its avoidance. Thus, the primary benefit of low leaf:sapwood allocation in progressively arid environments is to increase K_L and not to increase the contribution of stored water to transpiration.