Two-compartment method for determination of the oxygen transfer rate with electrochemical sensors based on sulfite oxidation

The dissolved oxygen concentration is a crucial parameter in aerobic bioprocesses due to the low solubility of oxygen in water. The present study describes a new method for determining the oxygen transfer rate (OTR) in shaken-culture systems based on the sodium sulfite method in combination with an electrochemical oxygen sensor. The method replaces the laborious titration of the remaining sulfite by an on-line detection of the end point of the reaction. This method is a two-step procedure that can be applied in arbitrary flasks that do not allow the insertion of electrodes. The method does not therefore depend on the type of vessel in which the OTR is detected. The concept is demonstrated by determination of the OTR for standard baffled 1-L shake flasks and for opaque Ultra Yield™ flasks. Under typical shaking conditions, k_La values in the standard baffled flasks reached values up to 220 h^-1, whereas the k_La values of the Ultra Yield flasks were significantly higher (up to 422 h^-1).     

A microwell platform for the scale-up of a multistep bioconversion to bench-scale reactors: Sitosterol side-chain cleavage

The microwell-scale approach is widely used for screening purposes and one-pot biotransformations, but it has seldom been applied to complex whole cell multistep bioconversions, requiring prolonged incubation periods. The present study aims to contribute to filling this gap. The side-chain cleavage of sitosterol to androstenedione (AD) with Mycobacterium sp. NRRL B-3805 cells was used as a model system, and focus was given to the screening of suitable bioconversion media with 24-well microwell plates. Results show that to perform this particular bioconversion growing cells are preferred over resting cells due to higher conversion yields obtained in aqueous medium. The use of resting cells may nevertheless present an interesting approach provided catalytic activity is retained throughout successive runs. Maintaining suitable aeration levels (air flow of 1 mL/min) allowed minimizing the decay of catalytic activity typically observed alongside consecutive bioconversion runs with resting cells. Microwell plates with dedicated oxygen and pH monitoring capabilities proved effective in media development for complex multistep bioconversions using relatively slow-growing bacteria. Under constant k_La (0.044/s) similar AD production and dissolved oxygen profiles were observed in microwell plates and in a bench-scale reactor. Selection of a suitable k_La value proved critical, since under lower k_La values scale-up proved unsuccessful. The same pattern was observed when other scale-up criteria were evaluated to perform the scale-up of this particular bioconversion. Results gathered seem to validate the proposed approach “from microwell plate to bench-scale fermentor”.     

Scaling‐up of complex whole‐cell bioconversions in conventional and non‐conventional media

The use of whole cells is becoming a more common approach in pharmaceutical and agrochemical industries in order to obtain pure compounds with fewer production steps, higher yields, and cleaner processes, as compared to those achieved with traditional strategies. Whole cells are often used as enzymes pools, in particular when multi‐step reactions and/or co‐factor regeneration are envisaged. Nonetheless, published information on the scale‐up of such systems both in aqueous and in two‐phase aqueous–organic systems is relatively scarce. The present work aims to evaluate suitable scale‐up criteria in conventional and non‐conventional medium for a whole‐cell bioconversion that uses resting cells of Mycobacterium sp. NRRL B‐3805 to cleave the side chain of β‐sitosterol, a poorly water‐soluble substrate. The experiments were performed in 24‐well microtiter plates and in 250 mL shaken flasks as orbital stirred systems, and in 300 mL stirred tanks as mechanically stirred systems. Results show that productivity yields were similar in all scales tested, when maintaining oxygen mass transfer coefficients constant in aqueous systems, or when maintaining constant volumetric power consumption in aqueous–organic two‐phase systems. Biotechnol. Bioeng. 2010;106: 619–626. © 2010 Wiley Periodicals, Inc.     

A novel method of simulating oxygen mass transfer in two-phase partitioning bioreactors

An empirical correlation, based on conventional forms, has been developed to represent the oxygen mass transfer coefficient as a function of operating conditions and organic fraction in two-phase, aqueous-organic dispersions. Such dispersions are characteristic of two-phase partitioning bioreactors, which have found increasing application for the biodegradation of toxic substrates. In this work, a critical distinction is made between the oxygen mass transfer coefficient, k(L)a, and the oxygen mass transfer rate. With an increasing organic fraction, the mass transfer coefficient decreases, whereas the oxygen transfer rate is predicted to increase to an optimal value. Use of the correlation assumes that the two-phase dispersion behaves as a single homogeneous phase with physical properties equivalent to the weighted volume-averaged values of the phases. The addition of a second, immiscible liquid phase with a high solubility of oxygen to an aqueous medium increases the oxygen solubility of the system. It is the increase in oxygen solubility that provides the potential for oxygen mass transfer rate enhancement. For the case studied in which n-hexadecane is selected as the second liquid phase, additions of up to 33% organic volume lead to significant increases in oxygen mass transfer rate, with an optimal increase of 58.5% predicted using a 27% organic phase volume. For this system, the predicted oxygen mass transfer enhancements due to organic-phase addition are found to be insensitive to the other operating variables, suggesting that organic-phase addition is always a viable option for oxygen mass transfer rate enhancement.     

A restructured framework for modeling oxygen transfer in two-phase partitioning bioreactors

This communication proposes a mechanistic modification to a recently published method for analyzing oxygen mass transfer in two-phase partitioning bioreactors (Nielsen et al., 2003), and corrects an oversight in that paper. The newly proposed modification replaces the earlier empirical approach, which treated the two liquid phases as a single, homogeneous liquid phase, with a two-phase mass transfer model of greater fundamental rigor. Additionally, newly developed empirical models are presented that predict the mass transfer coefficient of oxygen absorption in both aqueous medium and an organic phase (n-hexadecane) as a function of bioreactor operating conditions. Experimental values and theoretical predictions of mass transfer coefficients in two-phase dispersions, k(L)a(TP), are compared. The revised approach more clearly demonstrates the potential for oxygen mass transfer enhancement by organic phase addition, one of the motivations for employing a distinct second phase in a partitioning bioreactor.     

Scale up of a viscous fungal fermentation: application of scale-up criteria with regime analysis and operating boundary conditions

The scale up of the novel, pharmaceutically important pneumocandin (B(0)), from the filamentous fungus Glarea lozoyensis was successfully completed from pilot scale (0.07, 0.8, and 19 m(3)) to production scale (57 m(3)). This was accomplished, despite dissimilar reactor geometry, employing a combination of scale-up criteria, process sensitivity studies, and regime analysis using characteristic time constants for both oxygen mass transfer and bulk mixing. Dissolved oxygen tension, separated from the influence of agitation by gas blending at the 0.07 m(3)-scale, had a marked influence on the concentrations of pneumocandin analogs with different levels of hydroxylation, and these concentrations were used as an indicator of bulk mixing upon scale up. The profound impact of dissolved oxygen tension (DOT) (low and high levels) on analog formation dictated the use of constant DOT, at 80% air saturation, as a scale-up criterion. As a result k(L)a, Oxygen uptake rate (OUR) and hence the OTR were held constant, which were effectively conserved across the scales, while the use of other criterion such as P(g)/V(L), or mixing time were less effective. Production scale (57 m(3)) mixing times were found to be faster than those at 19 m(3) due to a difference in liquid height/tank diameter ratio (H(L)/D(T)). Regime analysis at 19 and 57 m(3) for bulk mixing (t(c)) and oxygen transfer (1/k(L)a) showed that oxygen transfer was the rate-limiting step for this highly shear thinning fermentation, providing additional support for the choice of scale-up criterion.     

Measuring kLa with randomly pulsed dynamic method

This reports on the determination of the overall oxygen transfer coefficient in a mechanically agitated vessel using a randomly pulsed dynamic method. This method consists in exciting the system by randomly switching the inlet gas stream with air or nitrogen with an identical volumetric flow rate. A pseudo-random binary sequence was used. This procedure is routinely used in process control for the identification of system's transfer function. The pulsed dynamic method gives good reliability (as compared with the traditional gassing-out method) and reproducibility in water. However, further improvement is needed before it can be used to monitor on-line the k(L)a during a fermentation.     

A shaken disposable bioreactor system for controlled insect cell cultivations at milliliter‐scale

While wave‐mixed and stirred bag bioreactors are common devices for rapid, safe insect cell culture‐based production at liter‐scale, orbitally shaken disposable flasks are mainly used for screening studies at milliliter‐scale. In contrast to the two aforementioned bag bioreactor types, which can be operated with standard or disposable sensors, shaker flasks have not been instrumented until recently. The combination of 250 mL disposable shake flasks with PreSens's Shake Flask Reader enables both pH and dissolved oxygen to be measured, as well as allowing characterization of oxygen mass transfer. Volumetric oxygen transfer coefficients (k_La‐values) for PreSens 250 mL disposable shake flasks, which were determined for the first time in insect cell culture medium at varying culture volumes and shaker frequencies, ranged between 4.4 and 37.9/h. Moreover, it was demonstrated that online monitoring of dissolved oxygen in shake flasks is relevant for limitation‐free growth of insect cells up to high cell densities in batch mode (1.6×10⁷ cells/mL) and for the efficient expression of an intracellular model protein.     

Development of a scale-up strategy for Chinese hamster ovary cell culture processes using the kLa ratio as a direct indicator of gas stripping conditions

Herein, we described a scale-up strategy focused on the dissolved carbon dioxide concentration (dCO₂) during fed-batch cultivation of Chinese hamster ovary cells. A fed-batch culture process for a 2000-L scale stainless steel (SS) bioreactor was scaled-up from similarly shaped 200-L scale bioreactors based on power input per unit volume (P/V). However, during the 2000-L fed-batch culture, the dCO₂ was higher compared with the 200-L scale bioreactor. Therefore, we developed an alternative approach by evaluating the k_La values of O₂ (k_La[O₂]) and CO₂ [k_La(CO₂)] in the SS bioreactors as a scale-up factor for dCO₂ reduction. The k_La ratios [k_La(CO₂)/k_La(O₂)] were different between the 200-L and 2000-L bioreactors under the same P/V condition. When the agitation conditions were changed, the k_La ratio of the 2000-L scale bioreactor became similar and the P/V value become smaller compared with those of the 200-L SS bioreactor. The dCO₂ trends in fed-batch cultures performed in 2000-L scale bioreactors under the modified agitation conditions were similar to the control. This k_La ratio method was used for process development in single-use bioreactors (SUBs) with shapes different from those of the SS bioreactor. The k_La ratios for the SUBs were evaluated and conditions that provided k_La ratios similar to the 200-L scale SS bioreactors were determined. The cell culture performance and product quality at the end of the cultivation process were comparable for all tested SUBs. Therefore, we concluded that the k_La ratio is a powerful scale-up factor useful to control dCO₂ during fed-batch cultures.