Device for sterile online measurement of the oxygen transfer rate in shaking flasks

The oxygen transfer rate (OTR) is the most suitable measurable parameter to quantify the physiological state of a culture of aerobic microorganisms since most metabolic activities depend on oxygen consumption. Online measurement of the oxygen transfer rate in stirred bioreactors is state of the art although technically difficult. However, the online determination of the oxygen transfer rate in shaking bioreactors under sterile conditions has not been possible until recently. A newly developed measuring device eliminates this deficit. Extremely useful information about cultivating conditions and the physiological state of microorganisms can be gained in early stages of research and bioprocess development from many reactors operated in parallel.     

Investigations of cephalosporin C production in an airlift tower loop reactor

Summary Cephalosporin C was produced by Cephalosporium acremonium in a 60 l airlift loop reactor on complex medium (with 30 kg/m³ peanut flour) in fed-batch operation. A final product concentration of 5 kg/m³ and a maximum productivity of 45 g/m³ h were attained. On-line analysis was used to determine ammonia, methionine, phosphate, reducing sugar and cephalosporin C by an autoanalyser, glucose by a flow injection analyser and cephalosporin C, penicillin N, deacetoxycephalosporin C, deacetylce-phalosporin C and methionine by HPLC. The volumetric productivity of the stirred tank reactor was higher than that of the airlift reactor because of differences in cell concentration. Specific productivities in relative to cell mass were similar in the two reactors. The substrate yield coefficient in the airlift reactor was twice that in the stirred tank reactor.Nomenclature E _o2 efficiency of oxygen transfer with regard to the specific power input - K _La volumetric mass transfer coefficient - OTR oxygen transfer rate - P power input - PR volumetric productivity of CPC - q _a volumetric aeration rate/broth volume (vvm) - SPR specific productivity with regard to RNA - V _L broth volume in reactor - z relative height of the aerated reactor     

Impact of nozzle operation on mass transfer in jet aerated loop reactors. Characterization and comparison to an aerated stirred tank reactor

The impact of mass transfer on productivity can become a crucial aspect in the fermentative production of bulk chemicals. For highly aerobic bioprocesses the oxygen transfer rate (OTR) and productivity are coupled. The achievable space time yields can often be correlated to the mass transfer performance of the respective bioreactor. The oxygen mass transfer capability of a jet aerated loop reactor is discussed in terms of the volumetric oxygen mass transfer coefficient k_La [h^?1] and the energetic oxygen transfer efficiency E [kgO₂ kW^?1 h^?1]. The jet aerated loop reactor (JLR) is compared to the frequently deployed aerated stirred tank reactor. In jet aerated reactors high local power densities in the mixing zone allow higher mass transfer rates, compared to aerated stirred tank reactors. When both reactors are operated at identical volumetric power input and aeration rates, local k_La values up to 1.5 times higher are possible with the JLR. High dispersion efficiencies in the JLR can be maintained even if the nozzle is supplied with pressurized gas. For increased oxygen demands (above 120 mmol L^?1 h^?1) improved energetic oxygen transfer efficiencies of up to 100 % were found for a JLR compared to an aerated stirred tank reactor operating with Rushton turbines.     

Estimation of Trichoderma QM 9414 biomass and growth rate by indirect means

Trichoderma QM 9414 was aerobically grown on glucose as the sole carbon and energy sources in a chemostat culture. The specific rates of glucose consumption (Q_G), oxygen consumption (Q), and carbon dioxide production (Q) at the steady state were measured to estimate the growth and maintenance requirements. From the results it was estimated that 2 mol adenosine triphosphate (ATP) were produced when1 mol NADH was oxidized through the respiratory chain of this microorganism. The true growth yield for ATP (Y_ATP) and specific ATP consumption rate for maintenance (Q) calculated with this value were 0.0106 g dry cell/mmol ATP and 5.2 mmol ATP/g dry cell/hr, respectively. Using the relationships between specific growth rate (μ) and (Q) and between μ and Q_G obtained from chemostat-culture data, cell and glucose concentration histories were estimated from the carbon dioxide production rate during the batch culture. The estimated cell concentrations agreed with the experimentally measured values. Glucose concentration were slightly overestimated.     

Oxygen transfer in three-phase airlift and stirred tank reactors using silicone oil as transfer vector

The use of organic liquids as vectors to enhance mass transfer has been applied since the 1970s. However, mass transfer in three-phase reactors is only partially understood. This paper aimed to characterize oxygen transfer in three-phase reactors containing air as gas, silicone oil as vector and water as aqueous phase. A mass transfer model that considers separately air/vector, vector/water and air/water oxygen transfers was developed. The model was used to describe oxygen transfer in airlift and stirred tank reactors containing from 0 to 50% of silicone oil. Under the experimental conditions, silicone oil had a positive effect on the overall oxygen transfer. In both reactor designs, the maximum overall oxygen transfer was observed with 10% silicone oil which was increased by 65 and 84% in the airlift and stirred reactor, respectively, compared to reactors operated without silicone oil. The overall transfer increase was mainly due to an enhanced air/water transfer. With 10% silicone oil, the air/water contribution to the overall oxygen transfer was 94.7 and 93.0% for the airlift and stirred reactor, respectively.     

Superior exercise performance in lifelong Tibetan residents of 4,400 m compared with Tibetan residents of 3,658 m

Few environments challenge human populations more than high altitude, since the accompanying low oxygen pressures (hypoxia) are pervasive and impervious to cultural modification. Work capacity is an important factor in a population's ability to thrive in such an environment. The performance of work or exercise is a measure of the integrated functioning of the O₂ transport system, with maximal O₂ uptake (VO) a convenient index of that function. Hypoxia limits the ability to transport oxygen: maximal O₂ uptake decreases with ascent to high altitude, and years of high altitude residence do not restore sea level VO values. Since Tibetans live and work at some of the highest altitudes in the world, their ability to exercise at very high altitude (<4,000 m) may define the limits of human adaptation to hypoxia. We transported 20 Tibetan lifelong residents of ≥4,400 m down to 3,658 m in order to compare them with 16 previously studied Tibetan residents of Lhasa (3,658 m). The two groups of Tibetans were matched for age, weight, and height. All studies were performed in Lhasa within 3 days of the 4,400 m Tibetans' arrival. Standard test protocol and criteria were used for attaining VO on a Monark bicycle ergometer, while measuring oxygen uptake (VO₂, ml/kg − min STPD), heart rate (bpm), minute ventilation (VE, 1/min BTPS), and arterial oxygen saturation (Sa, %). The 4,400 m compared with 3,658 m residents had, at maximal effort, similar VO₂ (48.5 ± 1.2 vs. 51.2 ± 1.4 ml/kg − min, P = NS), higher workload attained (211 ± 6 vs. 177 ± 7 watts, P < 0.01), lower heart rate (176 ± 2 vs. 191 ± 2 bpm, P < 0.01), lower ventilation (127 ± 5 vs. 149 ± 5 l/min BTPS, P < 0.01), and similar Sa(81.9 ± 1.0 vs. 83.7 ± 1.2%, P = NS). Furthermore, over the range of submaximal workloads, 4,400 m compared with 3,658 m Tibetans had lower VO₂ (P < 0.01), lower heart rates (P < 0.01), and lower ventilation (P < 0.01) and Sa (P < 0.05). We conclude that Tibetans living at 4,400 m compared with those residing at 3,658 m achieve greater work performance for a given VO₂ at submaximal and maximal workloads with less cardiorespiratory effort. Am J Phys Anthropol 105:21–31, 1998. © 1998 Wiley-Liss, Inc.     

Comparison of the performances of stirred tank and airlift tower loop reactors.

Following a consideration of the prerequisites for reactor comparison and the fundamental differences between stirred tank and airlift tower loop reactors, their performances are compared for the production of secondary metabolites: penicillin V by Penicillium chrysogenum, cephalosporin C by Cephalosporium acremonium, and tetracycline by Streptomyces aureofaciens. In stirred tank reactors, cell mass concentrations, volumetric productivities, and specific power inputs are higher than in airlift tower loop reactors. In the latter, efficiencies of oxygen transfer are higher, and specific productivities with regard to power input, substrate and oxygen consumptions, and yield coefficients of product formation with regard to substrate and oxygen consumptions are considerably higher than in stirred tank reactors. The prerequisites for improved performance are discussed.     

Oxygen limitation is a pitfall during screening for industrial strains

Oxygen supply is a key parameter in aerobic fermentation processes like the industrial production of amino acids. Although the oxygen transfer rate (OTR; or the volumetric oxygen transfer coefficient k_La) is routinely analyzed by engineers during stirred tank fermentations, it is often not taken into account by biologists conducting screening experiments in shake flasks. To show the importance of knowing how to avoid oxygen transfer limitations during primary screenings, Corynebacterium glutamicum ATCC 13032 (wild-type strain) and DSM 12866 (lysine-producing strain) were cultivated in shake flasks with different culture liquid volumes and under different shaking conditions. With the Respiration Activity Monitoring System, the OTR was determined quasi-continuously. Optical density as well as concentrations of lysine and byproducts (lactate, acetate, succinate) were determined off-line and correlated with the OTR signal. From the results, design criteria for improved screening in shaken bioreactors that help to avoid selection of suboptimal strains during early process development steps can be derived. Finally, the suitability of DSM 12866 as a strain for industrial processes with a high space–time yield is discussed.     

Jet aeration as alternative to overcome mass transfer limitation of stirred bioreactors

In industrial biotechnology increasing reactor volumes have the potential to reduce production costs. Whenever the achievable space time yield is determined by the mass transfer performance of the reactor, energy efficiency plays an important role to meet the requirements regarding low investment and operating costs. Based on theoretical calculations, compared to bubble column, airlift reactor, and aerated stirred tank, the jet loop reactor shows the potential for an enhanced energetic efficiency at high mass transfer rates. Interestingly, its technical application in standard biotechnological production processes has not yet been realized. Compared to a stirred tank reactor powered by Rushton turbines, maximum oxygen transfer rates about 200% higher were achieved in a jet loop reactor at identical power input in a fed batch fermentation process. Moreover, a model‐based analysis of yield coefficients and growth kinetics showed that E. coli can be cultivated in jet loop reactors without significant differences in biomass growth. Based on an aerobic fermentation process, the assessment of energetic oxygen transfer efficiency [kgO₂ kW^?1 h^?1] for a jet loop reactor yielded an improvement of almost 100%. The jet loop reactor could be operated at mass transfer rates 67% higher compared to a stirred tank. Thus, an increase of 40% in maximum space time yield [kg m^?3 h^?1] could be observed.     

Fed batch culture of Saccharomyces cerevisiae: A perspective of computer control to enhance the productivity in baker's yeast cultivation

A means to avoid the glucose effect in the production of baker's yeast from glucose and/or molasses in a fed batch culture by controlling the feed rate of fresh medium with an ad hoc measurement of the respiratory quotient, RQ, is presented. The feed rate is changed stepwise here such that the value of RQ ranges from 1.0 to 1.2 throughout the cultivation. Thus far, the specific growth rate based on the total cell mass and the growth yield obtained throughout are 0.24 hr^?1 and 0.55 g cell/g glucose. Prior to the experimental run mentioned above, equations to predetermine the feed rate and concentration of glucose in the feed are derived from the mass balance of limiting substrates (glucose). Since values of either RQ or I (Q x, oxygen consumption rate with respect to the total cell mass in the fermenter) can be measured quite easily and reliably, computer control of the fermentation in light of this information is discussed.     

Levels of DNA strand breaks and superoxide in phorbol ester-treated human granulocytes

Phorbol ester treatment of granulocytes triggers release of superoxide (O) and a concomitant burst of DNA strand breaks. The relationship between the amount of O and the number of DNA breaks has not previously been explored. To quantify the relatively large amount of O generated over a 40-min period by 1 × 10⁶ granulocytes/mL, a discontinuous “10-min pulse” method employing cytochrome c was used; 140 nmol O per 1 × 10⁶ cells was detected. DNA strand breaks were quantified by fluorimetric analysis of DNA unwinding (FADU). To vary the level of O released by cells, inhibitors of the respiratory burst were used. Sodium fluoride (1–10 mM) and staurosporine (2–10 nM) both inhibited O production. In both cases, however, inhibition of strand breakage was considerably more pronounced than inhibition of O. Zinc chloride (50–200 μM) inhibited both O and DNA breaks, approximately equally. Dinophysistoxin-1 (okadaic acid) inhibited O production more effectively than it inhibited DNA breaks. O dismutes to H₂O₂, a reactive oxygen species known to cause DNA breaks. The addition of catalase to remove extracellular H₂O₂ had no effect on DNA breakage. Using pulse field gel electrophoresis, few double-stranded breaks were detected compared to the number detected by FADU, indicating that about 95% of breaks were single-stranded. The level of DNA breaks is not directly related to the amount of extracellular O or H₂O₂ in PMA-stimulated granulocytes. We conclude that either an intracellular pool of these reactive oxygen species is involved in breakage or that the metabolic inhibitors are affecting a novel strand break pathway. J. Cell. Biochem. 66:219–228, 1997. © 1997 Wiley-Liss Inc.     

New milliliter‐scale stirred tank bioreactors for the cultivation of mycelium forming microorganisms

A novel milliliter‐scale stirred tank bioreactor was developed for the cultivation of mycelium forming microorganisms on a 10 milliliter‐scale. A newly designed one‐sided paddle impeller is driven magnetically and rotates freely on an axis in an unbaffled reaction vessel made of polystyrene. A rotating lamella is formed which spreads out along the reactor wall. Thus an enhanced surface‐to‐volume ratio of the liquid phase is generated where oxygen is introduced via surface aeration. Volumetric oxygen transfer coefficients (k_La) > 0.15 s^?1 were measured. The fast moving liquid lamella efficiently prevents wall growth and foaming. Mean power consumption and maximum local energy dissipation were measured as function of operating conditions in the milliliter‐scale stirred tank bioreactor (V = 10 mL) and compared to a standard laboratory‐scale stirred tank bioreactor with six‐bladed Rushton turbines (V = 2,000 mL). Mean power consumption increases with increasing impeller speed and shows the same characteristics and values on both scales. The maximum local energy dissipation of the milliliter‐scale stirred tank bioreactor was reduced compared to the laboratory‐scale at the same mean volumetric power input. Hence the milliliter impeller distributes power more uniformly in the reaction medium. Based on these data a reliable and robust scale‐up of fermentation processes is possible. This was demonstrated with the cultivation of the actinomycete Streptomyces tendae on both scales. It was shown that the process performances were equivalent with regard to biomass concentration, mannitol consumption and production of the pharmaceutical relevant fungicide nikkomycin Z up to a process time of 120 h. A high parallel reproducibility was observed on the milliliter‐scale (standard deviation < 8%) with up to 48 stirred tank bioreactors operated in a magnetic inductive drive. Rheological behavior of the culture broth was measured and showed a highly viscous shear‐thinning non‐Newtonian behavior. The newly developed one‐sided paddle impellers operated in unbaffled reactors on a 10 milliliter‐scale with a magnetic inductive drive for up to 48 parallel bioreactors allows for the first time the parallel bioprocess development with mycelium forming microorganisms. This is especially important since these kinds of cultivations normally exhibit process times of 100 h and more. Thus the operation of parallel stirred tank reactors will have the potential to reduce process development times drastically. Biotechnol. Bioeng. 2010; 106: 443–451. © 2010 Wiley Periodicals, Inc.     

Biotransformation of β-methyldigitoxin by cell cultures ofDigitalis lanata in airlift and stirred tank reactors

Summary Digitalis lanata cells were grown at dif-ferent dissolved-oxygen (DO) levels in 20-1 airlift reactors. A DO level of 30% saturation (using air for aeration) was found to be optimal for growth and the biotransformation ofβ-methyldigitoxin toβ-methyldigoxin. Product yield was further in-creased by using stirred tank reactors instead of the airlift reactor.     

Production of alginate by Azotobacter vinelandii grown at two bioreactor scales under oxygen-limited conditions

The oxygen transfer rate (OTR) was evaluated as a scale-up criterion for alginate production in 3- and 14-L stirred fermentors. Batch cultures were performed at different agitation rates (200, 300, and 600 rpm) and airflow rates (0.25, 0.5, and 1 vvm), resulting in different maximum OTR levels (OTR_max). Although the two reactors had a similar OTR_max (19 mmol L^?1 h^?1) and produced the same alginate concentration (3.8 g L^?1), during the cell growth period the maximum molecular weight of the alginate was 1,250 kDa in the 3-L stirred fermentor and 590 kDa in 14-L stirred fermentor. The results showed for the first time the evolution of the molecular weight of alginate and OTR profiles for two different scales of stirred fermentors. There was a different maximum specific oxygen uptake rate between the two fermenters, reaching 8.3 mmol g^?1 h^?1 in 3-L bioreactor and 10.6 mmol g^?1 h^?1 in 14-L bioreactor, which could explain the different molecular weights observed. These findings open the possibility of using $ q_{{{\text{O}}_{ 2} }} $ instead of OTR_max as a scaling criterion to produce polymers with similar molecular weights during fermentation.     

A kinetic analysis of hepatic microsomal activation of parathion and chlorpyrifos in control and phenobarbital-treated rats

A kinetic analysis of cytochrome P450-mediated desulfuration (activation) or dearylation (detoxication) showed that rat hepatic microsomes have a greater capacity to detoxify and a lower capacity to activate chlorpyrifos compared to parathion. Kinetic curves for the desulfuration of both parathion and chlorpyrifos were biphasic; K s of 0.23 and 71.3 μM were calculated for parathion, and 1.64 and 50.4 μM for chlorpyrifos. While phenobarbital (PB) exposure seemed to generally lower the K_mapp s for desulfuration except for the low K_m activity on chlorpyrifos, the results were not statistically significant. While the low K_m activity contributed 44 and 60% of the control V_max for parathion and chlorpyrifos, respectively, it contributed 50 and 17% in PB-treated rats. These studies have indicated the presence of a low K_m activity capable of functioning at very low substrate concentrations. A single dearylation K was calculated, 56.0 and 9.8 μM for parathion and chlorpyrifos, respectively. Phenobarbital exposure seemed to raise the Ks of dearylation; however, again, the results were not statistically significant. While numerous biochemical factors contribute to the overall toxicity levels of phosphorothionate insecticides, the in vitro efficiencies of hepatic microsomal desulfuration and dearylation of parathion and chlorpyrifos correspond to the acute toxicity levels.     

An Asymptotically Optimal Adaptive Selection Procedure in the Proportional Hazards Model with Conditionally Independent Censoring

Assume k independent populations are given which are distributed according to R, …,R (ϑ_i ∈ Θ ⊆ R ). Taking samples of size n the population with the smallest ϑ-value is to be selected. Using the framework of Le Cam's decision theory (Le Cam , 1986; Strasser , 1985) under mild regularity assumptions, an asymptotically optimal selection procedure is derived for the sequence of localized models. In the proportional hazards model with conditionally independent censoring, an asymptotically optimal adaptive selection procedure is constructed by substituting the unknown nuisance parameter by a kernel estimator.     

Bioreactor scale-up and oxygen transfer rate in microbial processes: An overview

In aerobic bioprocesses, oxygen is a key substrate; due to its low solubility in broths (aqueous solutions), a continuous supply is needed. The oxygen transfer rate (OTR) must be known, and if possible predicted to achieve an optimum design operation and scale-up of bioreactors. Many studies have been conducted to enhance the efficiency of oxygen transfer. The dissolved oxygen concentration in a suspension of aerobic microorganisms depends on the rate of oxygen transfer from the gas phase to the liquid, on the rate at which oxygen is transported into the cells (where it is consumed), and on the oxygen uptake rate (OUR) by the microorganism for growth, maintenance and production.     

Scale-up of naringinase production process based on the constant oxygen transfer rate for a novel strain of Bacillus methylotrophicus

Naringinase bioprocess based on Bacillus methylotrophicus was successfully scaled up based on constant oxygen transfer rate (OTR) as the scale-up criterion from 5-L bioreactor to 20-L bioreactor. OTR was measured in 5 and 20-L bioreactor under various operating conditions using dynamic method. The operating conditions, where complete dispersion was observed were identified. The highest OTR of 0.035 and 0.04?mMol/L/s was observed in 5 and 20-L bioreactor, respectively. Critical dissolved oxygen concentration of novel isolated strain B. methylotrophicus was found to be 20% of oxygen saturation in optimized medium. The B. methylotrophicus cells grown on sucrose had maximum oxygen uptake rate of 0.14?mMol/L/s in optimized growth medium. The cells produced the maximum naringinase activity of 751 and 778?U/L at 34?hr in 5 and 20-L bioreactors, respectively. The maximum specific growth rate of about 0.178/hr was observed at both the scales of operations. The maximum naringinase yield of 160 and 164?U/g biomass was observed in 5 and 20-L bioreactors, respectively. The growth and production profiles at both scales were similar indicating successful scale-up strategy for B. methylotrophicus culture.     

Intracerebral quantitative chromophore estimation from reflectance spectra captured during deep brain stimulation implantation

Quantification of blood fraction (f_blood), blood oxygenation (S), melanin, lipofuscin and oxidised and reduced Cytochrome aa 3 and c was done from diffuse reflectance spectra captured in cortex, white matter, globus pallidus internus (GPi) and subthalamus during stereotactic implantations of 29 deep brain stimulation (DBS) electrodes with the aim of investigating whether the chromophores can give physiological information about the targets for DBS. Double‐sided Mann‐Whitney U ‐tests showed more lipofuscin in GPi compared to white matter and subthalamus (p < 0.05). Compared to the other structures, f_bloodwas significantly higher in cortex (p < 0.05) and S lower in GPi (p < 0.05). Median values and range for f_blood were 1.0 [0.2–6.0]% in the cortex, 0.3 [0.1–8.2]% in white matter, 0.2 [0.1–0.8]% in the GPi and 0.2 [0.1–11.7]% in the subthalamus. Corresponding values for S was 20 [0–81]% in the cortex, 29 [0–78]% in white matter, 0 [0–0]% in the GPi and 0 [0–92]% in the subthalamus. In conclusion, the measurements indicate very low oxygenation and blood volume for DBS patients, especially in the GPi. It would be of great interest to investigate whether this is due to the disease, the normal situation or an artefact of doing invasive measurements. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Development of a new commercial-scale airlift fermentor for rapid growth of yeast

To grow yeast rapidly, it is necessary to supply sufficient oxygen to the yeast and to effectively remove the heat of the fermentation. We succeeded in developing a commercial-scale fermentor for growing a food yeast (Candida utilis) to produce RNA rapidly. This fermentor is an internal-loop airlift type with vertical heat transfer tubes between inner and the outer columns. The volume of the fermentor is 145 m³ (working volume 75 m³). The oxygen transfer rate (OTR) was 9.9 kg-O₂/m³/h using a superficial gas velocity of 30 cm/s based on the outer column. Much of the heat of fermentation and the energy resulting from aeration could be removed effectively by the heat transfer tubes. This unique airlift fermentor was driven at a dilution rate of 0.43 h⁻¹ for about 70 d, with the yeast concentration being maintained at 22.8 kg-dry cell/m³. The yeast production rate was 9.79 kg-dry cell/m³/h. Compared with a traditional stirred-type fermentor, two Vogelbush-type fermentors and another airlift fermentor, our fermentor was far superior with respect to OTR and yeast productivity.