A system of miniaturized stirred bioreactors for parallel continuous cultivation of yeast with online measurement of dissolved oxygen and off‐gas

Chemostat cultivation is a powerful tool for physiological studies of microorganisms. We report the construction and application of a set of eight parallel small‐scale bioreactors with a working volume of 10 mL for continuous cultivation. Hungate tubes were used as culture vessels connected to multichannel‐peristaltic pumps for feeding fresh media and removal of culture broth and off‐gas. Water saturated air is sucked into the bioreactors by applying negative pressure, and small stirrer bars inside the culture vessels allow sufficient mixing and oxygen transfer. Optical sensors are used for non‐invasive online measurement of dissolved oxygen, which proved to be a powerful indicator of the physiological state of the cultures, particularly of steady‐state conditions. Analysis of culture exhaust‐gas by means of mass spectrometry enables balancing of carbon. The capacity of the developed small‐scale bioreactor system was validated using the fission yeast Schizosaccharomyces pombe, focusing on the metabolic shift from respiratory to respiro‐fermentative metabolism, as well as studies on consumption of different substrates such as glucose, fructose, and gluconate. In all cases, an almost completely closed carbon balance was obtained proving the reliability of the experimental setup. Biotechnol. Bioeng. 2013; 110: 535–542. © 2012 Wiley Periodicals, Inc.     

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.     

Multiplying steady-state culture in multi-reactor system

Cultivation of microorganisms in batch experiments is fast and economical but the conditions therein change constantly, rendering quantitative data interpretation difficult. By using chemostat with controlled environmental conditions the physiological state of microorganisms is fixed; however, the unavoidable stabilization phase makes continuous methods resource consuming. Material can be spared by using micro scale devices, which however have limited analysis and process control capabilities. Described herein are a method and a system combining the high throughput of batch with the controlled environment of continuous cultivations. Microorganisms were prepared in one bioreactor followed by culture distribution into a network of bioreactors and continuation of independent steady state experiments therein. Accelerostat cultivation with statistical analysis of growth parameters demonstrated non-compromised physiological state following distribution, thus the method effectively multiplied steady state culture of microorganisms. The theoretical efficiency of the system was evaluated in inhibitory compound analysis using repeated chemostat to chemostat transfers.     

Optical method for the determination of the oxygen-transfer capacity of small bioreactors based on sulfite oxidation.

The growth of microorganisms may be limited by operating conditions which provide an inadequate supply of oxygen. To determine the oxygen-transfer capacities of small-scale bioreactors such as shaking flasks, test tubes, and microtiter plates, a noninvasive easy-to-use optical method based on sulfite oxidation has been developed. The model system of sodium sulfite was first optimized in shaking-flask experiments for this special application. The reaction conditions (pH, buffer, and catalyst concentration) were adjusted to obtain a constant oxygen transfer rate for the whole period of the sulfite oxidation reaction. The sharp decrease of the pH at the end of the oxidation, which is typical for this reaction, is visualized by adding a pH dye and used to measure the length of the reaction period. The oxygen-transfer capacity can then be calculated by the oxygen consumed during the complete stoichiometric transformation of sodium sulfite and the visually determined reaction time. The suitability of this optical measuring method for the determination of oxygen-transfer capacities in small-scale bioreactors was confirmed with an independent physical method applying an oxygen electrode. The correlation factor for the maximum oxygen-transfer capacity between the chemical model system and a culture of Pseudomonas putida CA-3 was determined in shaking flasks. The newly developed optical measuring method was finally used for the determination of oxygen-transfer capacities of different types of transparent small-scale bioreactors.     

Scale-up strategy for bioreactors with Newtonian and non-Newtonian broths

In the scale-up of bioreactors one of the most commonly used criteria for aerobic processes is to keep the oxygen transfer coefficient constant. Nevertheless, the reproduction of the behaviour of a cell population at different scales is not dependent just on the oxygen transfer capability but rather on the oxygen transfer rate, that must be high enough to cope with the oxygen demand of the culture. In this work a strategy of scale-up is proposed based in geometric similarity, constant impeller tip speed, and constant oxygen transfer rate. The effects of the dissolved oxygen concentration, the scale-up ratio and the rheology of the broth are analysed.     

Performance of airlift bioreactors in the cultivation of some antibiotic producing microorganisms

The specific aspects of airlift reactors emphasizing their function relevance to particular application as bioreactors are presented. The two main groups of airlift reactors – external-loop and concentric-tube reactors – were investigated on a pilot-plant scale with regard to their performance during the cultivation of unicellular and filamentous microorganisms which produce Bacitracin, Cephalosporin C and Nystatin. Some results were compared to those obtained in conventional stirred tank bioreactors. The comparison was carried out based on physical properties (oxygen transfer rate (OTR), volumetric mass transfer coefficient (k_La) and efficiency of oxygen transfer (E)), cell mass, productivity and substrate consumption, secondary metabolite production, and efficiency of the product formation with regard to the specific power input. It was shown that B. licheniformis, C. acremonium and S. noursei fermentations occurred similarly to those performed in stirred vessels, proving that the capacity of the airlift bioreactors surpassed the problems which arise from the morphology and rheology of the broths. From the chemical engineering point of view, it was obvious that the primary tasks of a bioreactor (uniform distribution of microorganisms and nutrients over the entire fermenter volume, appropriate supply of biomass with nutrients and oxygen) were fulfilled by the airlift bioreactors tested. In addition, the efficiency of oxygen transfer (OTR referred to power input) in the airlift fermenters proved to be about 38% higher than in the stirred tank bioreactors (expressed as average values), while the sorption efficiency (OTR referred to antibiotic production) was found to be 22% greater in the airlift system than in an STR. Therefore, the biosyntheses were performed with about a 30–40% increase in energy efficiency and energy savings compared to the conventional system. Moreover, the lack of mechanical devices in the airlift system provides greater safety and a gentler environment for the cultivation of microorganisms.     

Description and evaluation of reciprocating plate bioreactors

The environment in which live microorganisms has a major impact on their productivity. One important factor is the mechanical mixing that is used to promote good heat and mass transfer in bioreactors. In this paper, the performance of reciprocating plate bioreactors is first evaluated for their ability to produce high oxygen transfer coefficients. Pure water and a glycerol water (5050 wt%) solution are used for this evaluation. Then, the performance of reciprocating plate bioreactors for the production of an exocellular polysaccharide (pullulan) by yeast Aureobasidium pullulans is analyzed in terms of quantity and quality of the polysaccharide. Results clearly show that a more efficient substrate utilisation is achieved with reciprocating plate bioreactors.     

Analyzing the suitability of a baffled orbitally shaken bioreactor for cells cultivation using the computational fluid dynamics approach

Orbitally shaken bioreactors (OSRs) is one of important bioreactors for mammalian cells cultivation in suspension, especially for the screening of valuable microorganisms and in basic bioprocess development experiments. However, the suitability of OSRs for cells culture in large scale is still under development. In this article, a new kind of OSRs with baffle structure was proposed and a three-dimensional CFD model was established to analyze the influence of baffle structure on the flow field. Lower installation height of baffles was found suitable for improving the mixing efficiency. Compared to the unbaffled OSR, the baffled OSR could enhance the level of oxygen transfer largely but the oxygen transfer rate was independent on the baffle installation height. Moreover, as the baffle installation height increased, the energy transferred for liquid motion was decreased. Finally, the shear stress of the baffled OSRs proposed was gentle for mammalian cells growth. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2746, 2019     

Shear conditions in clavulanic acid production by Streptomyces clavuligerus in stirred tank and airlift bioreactors

In biochemical processes involving filamentous microorganisms, the high shear rate may damage suspended cells leading to viability loss and cell disruption. In this work, the influence of the shear conditions in clavulanic acid (CA) production by Streptomyces clavuligerus was evaluated in a 4-dm(3) conventional stirred tank (STB) and in 6-dm(3) concentric-tube airlift (ALB) bioreactors. Batch cultivations were performed in a STB at 600 and 800 rpm and 0.5 vvm (cultivations B1 and B2) and in ALB at 3.0 and 4.1 vvm (cultivations A1 and A2) to define two initial oxygen transfer conditions in both bioreactors. The average shear rate ([Formula: see text]) of the cultivations was estimated using correlations of recent literature based on experimental data of rheological properties of the broth (consistency index, K, and flow index, n) and operating conditions, impeller speed (N) for STB and superficial gas velocity in the riser (UGR) for ALB. In the same oxygen transfer condition, the [Formula: see text] values for ALB were higher than those obtained in STB. The maximum [Formula: see text] presented a strong correlation with a maximum consistency index (K (max)) of the broth. Close values of maximum CA production were obtained in cultivations A1 and A2 (454 and 442 mg L(-1)) with similar maximum [Formula: see text] values of 4,247 and 4,225 s(-1). In cultivations B1 and B2, the maximum CA production of 269 and 402 mg L(-1) were reached with a maximum [Formula: see text] of 904 and 1,786 s(-1). The results show that high values of average shear rate increase the CA production regardless of the oxygen transfer condition and bioreactor model.     

Evaluation of oxygen transfer rates in stirred-tank bioreactors for clinical manufacturing

Several methods are available for determining the volumetric oxygen transfer coefficient in bioreactors, though their application in industrial bioprocess has been limited. To be practically useful, mass transfer measurements made in nonfermenting systems must be consistent with observed microbial respiration rates. This report details a procedure for quantifying the relationship between agitation frequency and oxygen transfer rate that was applied in stirred-tank bioreactors used for clinical biologics manufacturing. The intrinsic delay in dissolved oxygen (DO) measurement was evaluated by shifting the bioreactor pressure and fitting a first-order mathematical model to the DO response. The dynamic method was coupled with the DO lag results to determine the oxygen transfer rate in Water for Injection (WFI) and a complete culture medium. A range of agitation frequencies was investigated at a fixed air sparge flow rate, replicating operating conditions used in Pichia pastoris fermentation. Oxygen transfer rates determined by this method were in excellent agreement with off-gas calculations from cultivation of the organism (P = 0.1). Fermentation of Escherichia coli at different operating parameters also produced respiration rates that agreed with the corresponding dynamic method results in WFI (P = 0.02). The consistency of the dynamic method results with the off-gas data suggests that compensation for the delay in DO measurement can be combined with dynamic gassing to provide a practical, viable model of bioreactor oxygen transfer under conditions of microbial fermentation.     

Application of bioreactor design principles to plant micropropagation

Principles of oxygen consumption, oxygen transport, suspension, and mixing are discussed in the context of propagating aggregates of plant tissue in liquid suspension bioreactors. Although micropropagated plants have a relatively low biological oxygen demand (BOD), the relatively large tissue size and localization of BOD in meristematic regions will typically result in oxygen mass transfer limitations in liquid culture. In contrast to the typical focus of bioreactor design on gas–liquid mass transfer, it is shown that media-solid mass transfer limitations limit oxygen available for aerobic plant tissue respiration. Approaches to improve oxygen availability through gas supplementation and bioreactor pressurization are discussed. The influence of media components on oxygen availability are also quantified for plant culture media. Experimental studies of polystyrene beads in suspension in a 30-l air-lift and stirred bioreactors are used to illustrate design principles for circulation and mixing. Potential limitations to the use of liquid suspension culture due to plant physiological requirements are acknowledged.     

Model analysis of biological oxygen transfer enhancement in surface-aerated bioreactors

A model was developed to evaluate the effects of cells and surfactants on oxygen transfer in surface-aerated bioreactors. The model assumed the presence of serial layers of adsorbed surfactants and microorganisms directly adjacent to the gas-liquid interface due to their surface activities, followed by a stagnant liquid layer to account for the oxygen transfer resistance in the liquid phase. The interfacial surfactant film, although posing as an additional resistance, was found to have negligible effect on the oxygen transfer rate because of its extremely small thickness as compared to the cell monolayer and the stagnant liquid layer. On the other hand, cells affect oxygen transfer by two mechanisms: the biological enhancement due to the respiration of interfacial cells and the physical blocking resulting from the semipermeable nature of cell bodies. Due to the low specific oxygen uptake rates of the sludges, the two mechanisms were found to be of comparable importance in activated-sludge systems; the oxygen transfer enhancement factor, E, varied from about 0.97 to 1.10 depending on the operating conditions. The biological enhancement effect, however, predominated in fermentations of actively growing bacteria. At relatively low agitation speed (e. g., 300 rpm), the value of E could reach about 3 to 5 in fermentations with high cell concentrations. Effects of other operating variables, such as the agitation intensity, the oxygen content in the mixed liquor, and the bulk cell concentration, on biological oxygen transfer enhancement were also studied. (c) 1992 John Wiley & Sons, Inc.     

Introduction to advantages and problems of shaken cultures

Shaking bioreactors are the most frequently used reaction vessels in biotechnology and have been so for many decades. In spite of their large practical importance, very little is known about the characteristic properties of shaken cultures from an engineering point of view. The few publications available contain to some extent contradicting statements and conflicting advice concerning the correct operating conditions of shaking bioreactors. Depending on the investigated microbial system, the engineering parameters may more or less significantly influence the experimental results in a quantitative as well as in a qualitative manner. Unfortunately, these kind of interactions are often overlooked or ignored by scientists. Precise knowledge about the controlling hydrodynamic phenomena in shaking bioreactors and quantitative information about the physical parameters influencing the cultures are needed to assure reproducible and meaningful operating conditions. In this introduction, the state of the art of culturing microorganisms in shaking bioreactors is reviewed and some issues of their practical application in screening and process development projects are addressed.     

Effects of the oxygen transfer rate on ferrous iron oxidation by Thiobacillus ferrooxidans

Ferrous iron oxidation by Thiobacillus ferrooxidans was studied in shake flasks and a bubble column under different aeration conditions. The maximum biooxidation rate constant was affected by oxygen transfer only at low aeration intensities. At oxygen transfer rates higher than 0.03 mmol O₂ l⁻¹ min⁻¹, the maximum biooxidation rate constant was about 0.050 h⁻¹ in both shake flasks of different size and the bubble column. The oxygen transfer rate could be used as a basis for scaling up bioreactors for ferrous iron biooxidation by T. ferrooxidans.     

Scale‐up analysis for a CHO cell culture process in large‐scale bioreactors

Bioprocess scale‐up is a fundamental component of process development in the biotechnology industry. When scaling up a mammalian cell culture process, it is important to consider factors such as mixing time, oxygen transfer, and carbon dioxide removal. In this study, cell‐free mixing studies were performed in production scale 5,000‐L bioreactors to evaluate scale‐up issues. Using the current bioreactor configuration, the 5,000‐L bioreactor had a lower oxygen transfer coefficient, longer mixing time, and lower carbon dioxide removal rate than that was observed in bench scale 5‐ and 20‐L bioreactors. The oxygen transfer threshold analysis indicates that the current 5,000‐L configuration can only support a maximum viable cell density of 7 × 10⁶ cells mL^?1. Moreover, experiments using a dual probe technique demonstrated that pH and dissolved oxygen gradients may exist in 5,000‐L bioreactors using the current configuration. Empirical equations were developed to predict mixing time, oxygen transfer coefficient, and carbon dioxide removal rate under different mixing‐related engineering parameters in the 5,000‐L bioreactors. These equations indicate that increasing bottom air sparging rate is more efficient than increasing power input in improving oxygen transfer and carbon dioxide removal. Furthermore, as the liquid volume increases in a production bioreactor operated in fed‐batch mode, bulk mixing becomes a challenge. The mixing studies suggest that the engineering parameters related to bulk mixing and carbon dioxide removal in the 5,000‐L bioreactors may need optimizing to mitigate the risk of different performance upon process scale‐up. Biotechnol. Bioeng. 2009;103: 733–746. © 2009 Wiley Periodicals, Inc.     

Enhancing oxygen transfer in surface-aerated bioreactors by stable foams.

To enhance oxygen transfer in surface-aeration bioreactors, stabilized foams were generated to increase the gas-liquid interfacial area by slowly introducing coarse bubbles into media containing fetal bovine serum. The bubble sparging rates were so low (i.e., 20 and 50 mL/h) that the contribution to oxygen transfer from these bubbles was due to foaming instead of bubbling. Furthermore, no physical cell damage caused by bubble sparging was observed. Oxygen transfer coefficients, kLa, in the bioreactors were measured in cell-free media. Without the foam-stabilizing agent (i.e., serum), no appreciable change in kLa was observed due to the bubble sparging. On the other hand, with serum, kLa increased with increasing serum content and bubble sparging rate and corresponded well with the degree of foaming. With 10% fetal bovine serum and a bubble sparging rate of 50 mL/h, kLa increased approximately 90% compared with no foaming. The enhancing effect of foam on oxygen transfer in surface aeration bioreactors has been further demonstrated with hybridoma cultures simultaneously grown in three identical bioreactors with and without stabilized foams.     

Microbial physiological state at low growth rate in natural and engineered ecosystems

Microbes often grow in nature and bioreactors at very low growth rates. However, the physiological consequences at low growth rates have not been explored as completely as at faster growth rates or under starvation conditions. Nutrient flux to (and through) the cell surface and non-growth-dependent energy consumption (maintenance) are important considerations under these conditions. The biomass recycle reactor is a system to explore physiological state at low growth rate, and to optimize certain industrial process rates.     

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.     

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.     

Microscopic green algae and cyanobacteria in high-frequency intermittent light

The effects of fluctuations in the irradiance onScenedesmus quadricauda, Chlorella vulgaris andSynechococcus elongatus were studied in dilute cultures using arrays of red light emitting diodes. The growth rate and the rate of photoinhibition were compared using intermittent and equivalent continuous light regimes in small-size (30 ml) bioreactors. The CO₂ dependent photosynthetic oxygen evolution rates in the intermittent and continuous light regimes were compared for different light/dark ratios and different mean irradiances. The kinetics of the electron transfer reactions were investigated using a double-modulation fluorometer. The rates of photosynthetic oxygen evolution normalized to equal mean irradiance were lower or equal in the intermittent light compared to the maximum rate found in the equivalent optimal continuous light regime. In contrast, the growth rates in the intermittent light can be higher than the growth rate in the equivalent continuous light. Photoinhibition is presented as an example of a physiological process affecting the growth rate that occurs at different rates in the intermittent and equivalent continuous lights. The difference in the dynamics of the redox state of the plastoquinone pool is proposed to be responsible for the low photoinhibition rates observed in the intermittent light.