Volumetric oxygen transfer coefficients (kLa) in batch cultivations involving non-Newtonian broths

doi:10.1016/S1369-703X(01)00092-4

Biochemical Engineering Journal

Volume 8, Issue 2, September 2001, Pages 111-119

https://doi.org/10.1016/S1369-703X(01)00092-4 Get rights and content

Abstract

The oxygen transfer in non-Newtonian fermentation broths of Aspergillus awamori, during batch cultivations in conventional 10 l bioreactor has been investigated. Values of the volumetric oxygen transfer coefficient (k_La), obtained at various impeller speeds, air flow rates and at distinct initial substrate concentrations were correlated with operational variables, geometric parameters of the system and physical properties of the broths utilising rigorous techniques in order to obtain a set of reliable and accurate data. An experimental device was constructed for on-line rheological measurements, and the apparent dynamic viscosity was determined from the broth rheograms. In order to measure power requirements, a torque meter was developed and non-Newtonian fluids with rheological characteristics similar to the Aspergillus fermented broths were utilised to obtain reference curves and correlations in the fermentor where the cultivations took place. Gas balancing method and a modified dynamic method were utilised simultaneously to determine k_La values. The rigorous methods thus developed allowed adequate evaluation of the oxygen transfer in the cultivations and also permitted good fits of four different traditional correlations for k_La to the experimental data.

Introduction

Fermentation broths containing mycelial cells frequently exhibit a pseudoplastic non-Newtonian rheological behaviour, which can be described by the power-law model. This behaviour exerts a profound effect on the bioreactor performance, affecting mixing pattern, power requirement, heat and mass transfer processes [1]. The increase in the broth apparent viscosity (μ_ap) during aerobic fermentations can be partially compensated by increments in the operating conditions (N and Q), in order to maintain adequate k_La values. Nevertheless, high impeller speeds (N) lead to the formation of high shear zones close to the impellers, with consequent physical damage to the cells and a reduction in the process productivity [2]. Due to the importance of the volumetric oxygen transfer coefficient (k_La) in the performance and scale-up of conventional bioreactors, the literature describing various correlations for k_La in Newtonian fluids is rather extensive. However, particular care should be taken when applying these correlations to non-Newtonian systems containing electrolytes such as fermentation broths [3].

Two types of correlations have been proposed for the volumetric oxygen transfer coefficient (k_La). The first does not make use of any dimensional criterion. In these correlations, k_La is related to the gassed power consumption per unit volume of broth (P_g/V) and the superficial gas velocity (v_s), as originally proposed by Cooper et al. [4]: $k_{L} a α P_{g} V^{a_{1}} (v_{s})^{b_{1}}$ where the values of the constants a₁ and b₁ may vary considerably, depending on the system geometry, the range of variables covered and the experimental methodology used. Although initially developed for fluids very distinct from fermentation broths, this type of correlation has been widely used in fermentation systems [3], [5], [6], [7]. In a more recent work, Montes et al. [8] determined values of k_La in yeast broths (Trigonopsis variabilis) over wide ranges of both impeller speeds and superficial gas velocities in three different mechanically-stirred, sparger-aerated and baffled bioreactors (2, 5 and 15 l) in order to consider the effect of the fermentor scale-up on k_La. Experimental data were fitted using the correlation proposed by Cooper et al. [4] and the values for the parameters a₁, b₁ and proportionality constant were 0.35, 0.41 and 3.2×10⁻³, respectively. Due to the fact that most of the yeast broths behave as slightly non-coalescent fluid, according to the authors, the correlation improved the prediction of k_La values with respect to other generic correlations usually developed for strong coalescent and non-coalescent fluids. Extended forms of this first type of relationship have been proposed in the literature, incorporating terms like impeller speed (N) and the fluid apparent viscosity (μ_ap). For example, Ryu and Humphrey [9] observed the influence of the broth apparent viscosity (μ_ap) on k_La in penicillin fermentation and proposed the following correlation: $k_{L} a α P_{g} V^{a_{2}} (v_{s})^{b_{2}} (μ_{ap})^{c_{2}}$ where again, the constants a₂, b₂, c₂ as well as the proportionality constant, depend on the geometry of the system. More recently, Garcı́a-Ochoa and Gómez [10] utilised this correlation to fit experimental data obtained in a stirred tank reactor of 20 l of working volume. The rheology of the xanthan gum solutions was described both in terms of the power-law and the Casson models. Experiments were performed considering the effect of the number and type of stirrers (paddle or turbine), the number of blades of the stirrers and the sparger type (ring and disk) in the volumetric oxygen transfer coefficient (k_La). In the same way, Gavrilescu et al. [1] have assessed the problem of oxygen transfer in antibiotic broths produced by nine different microorganisms belonging to the actinomycetes and the fungi. The following equation for k_La was established from a wide variety of experimental data obtained in 20 and 100 m³ stirred tank bioreactors: $k_{L} a=0.025 P_{g} V^{0.4} (v_{s})^{0.5} (N)^{0.5} μ_{ap} μ_{g}^{0.65}$ Additionally, Shin et al. [11] verified that in high cell density cultures of fast-growing aerobes such as recombinant E. coli where the cell mass increases up to more than 70 g/l, the oxygen availability can be the rate-limiting of the fermentation process. In that way, the following correlation for k_La incorporating the effect of cell density (X) in oxygen transfer has been proposed: $k_{L} a=0.0192 P_{g} V^{0.55} (v_{s})^{0.64} (1+2.12X+0.20X^{2})^{−0.25}$ The second type of correlation for k_La is based on dimensional analysis. This approach presents certain advantages because the correlations obtained for a known system can be used to estimate k_La in other systems with different dimensions. Yagi and Yoshida [12] proposed the following correlation, valid for both Newtonian and non-Newtonian fluids, for the standard six-bladed turbine in standard configuration in a vessel of 0.25 m diameter: $Sh^{∗} =0.060(Re_{m})^{1.50} (Sc)^{0.50} (Fr)^{0.19} μ_{ap} v_{s} σ^{0.60} ND_{i} v_{s}^{0.32}$ This correlation fitted to the experimental data in a reasonable extent, with maximum deviations of 30 and 80%, between experimental and calculated Sh^∗ values, for Newtonian and non-Newtonian fluids, respectively. In order to determine the combined effects of the variables, including operational conditions, liquid and gas properties and geometry of the tank, Garcı́a-Ochoa and Gómez [10] employed a similar dimensionless equation to fit the set of experimental data of k_La obtained in the same system under operational conditions above specified. Li et al. [13] proposed a relationship among k_La, the impeller speed (N) and the apparent viscosity (μ_ap), for fermentation broths of Aureobasidium pullulans and Xanthomonas campestris in 100 l fermentor, which correlated quite well the great variety of experimental data, as follows, respectively: $Sh^{∗} =5.40×10^{4} (Fr)^{0.45} μ_{ap} μ_{w}^{−0.33}$ $Sh^{∗} =3.32×10^{3} (Fr)^{0.23} μ_{ap} μ_{w}^{−0.58}$ Zlokarnik [14] suggested, also through dimensional analysis, a general dependence of k_La on physico-chemical properties and operating conditions, as follows: $k_{L} a Q V_{i}^{−1} α P_{g_{i}} Q^{∗}^{a_{3}} (Sc)^{b_{3}} (σ^{∗})^{c_{3}} (Si^{∗})^{d_{3}}$ where the dimensionless variable Si^∗, not exactly defined, is related to the coalescence behaviour of solutions. However, this correlation presents a feature not found in the others since the variable P_{g_i} represents the gassed power consumption per impeller, this equation can be applied to systems with different numbers of impellers and, therefore, with different geometries. This possibility was verified by Jurecic et al. [3], investigating submerged cultures of Bacillus licheniforms for bacitracin production in bioreactors of 100 l and 67.5 m³ with two and four turbine impellers, respectively.

Although several correlations have been developed for different systems, most of them are not specific to fermentation broths or, when developed with such a purpose, they do not determine all the variables throughout the cultivation. Surface tension of the liquid (σ), for instance, is considered to be constant throughout the whole cultivation and, therefore, it is not considered in the correlations. Furthermore, in the development of k_La correlations in cultivations involving filamentous microorganisms, particular care should be taken when measuring some variables, like the rheological properties of the broth and the k_La itself, when traditional equipment and methodologies are utilised. In recent works, Badino Jr. et al. [15], as well as Svihla et al. [16], verified that the use of conventional bench rheometers, such as the concentric cylinder, to determine rheological properties of viscous mycelial suspensions, is often unsatisfactory. The main problems are caused by pellet’s size, generally of the same order of magnitude as the annulus, as well as the tendency of the suspension to become heterogeneous due to settling and particle interaction. Thus, they propose the use of rotational rheometer provided with a helical impeller to obtain reliable and accurate rheological data for filamentous fermentation broths. In addition, Olsvik and Kristiansen [17] concluded that on-line continuous measurement of broth rheology enables the uniform treatment of the samples and statistically more “correct” measurements.

The aim of this work is to investigate the influence of operating conditions on the rheological properties of the fermented broth and on the oxygen transfer during batch cultivations of A. awamori in a bench scale fermentor. Methodologies for on-line rheological measurements and for determination of both power consumption and k_La have been developed in order to obtain a set of reliable and accurate data. Four classical correlations mentioned previously (, , , ) for the volumetric oxygen transfer coefficient (k_La), including the operating conditions, geometrical parameters of the system and physical properties of the broth (apparent viscosity and also surface tension of the filtrated broth), were fitted to the experimental values.

Section snippets

Microorganism and culture media

A spore suspension (2×10⁷ spores per millilitre) of A. awamori NRRL 3112, stored in cryotubes (glycerol 20% v/v) at −20°C, was used throughout this work.

Growth medium was composed of (in g/l): total reducing sugars (TRS) from cassava flour and assayed as glucose, 20.0; yeast extract, 0.2; MgSO₄·7H₂O, 1.0; (NH₄)₂SO₄, 10.0; Na₂HPO₄·12H₂O, 7.56; KH₂PO₄, 7.0, pH 5.0. Two fermentation media with different compositions were utilised. The first had the same composition as the growth medium and the

Rheological behaviour of fermentation broths

With the average shear rate (γ_av) ranging from 0.96 to 72.27 s⁻¹, all fermentation broths at different cultivation times, behaved as non-Newtonian pseudoplastic fluids. Good fits between the power law model and the experimental data were achieved, showing that on-line rheological measurement is a consistent and reliable technique for mycelial cultivations. Fig. 2 illustrates typical flow curves (rheograms) obtained at different times for the run E40-7-6. Significant changes in the rheological

Conclusions

The main objective of this work was to investigate the influence of operating conditions on the rheological properties of the broth and on the oxygen transfer during batch cultivations of A. awamori in a bench scale fermentor. Four classical correlations found in the literature, which correlate the volumetric oxygen transfer coefficient (k_La) with the process operational variables (N, Q and P_g) and the geometric parameters of the system has been utilised. In particular, in some of these

References (29)

F.J. Montes et al.
Proc. Biochem.
(1999)
F. Garcı́a-Ochoa et al.
Biochem. Eng. J.
(1998)
G.-Q. Li et al.
J. Chem. Tech. Biotechnol.
(1995)
E.S. Olsvik et al.
Biotechnol. Adv.
(1994)
M. Gavrilescu et al.
Acta Biotechnol.
(1993)
J.J. Smith et al.
Biotechnol. Bioeng.
(1990)
R. Jurecic et al.
The Can. J. Chem. Eng.
(1984)
C.M. Cooper et al.
Ind. Eng. Chem.
(1944)
M. Griot, V. Saner, E. Heinzle, I.J. Dunn, J.R. Bourne, in: Proceedings of the 2nd International Conference on...
V. Linek et al.
Biotechnol. Bioeng.
(1990)

A.C. Badino Jr., M. Barboza, C.O. Hokka, Advances in Bioprocess Engineering, Kluwer Academic Publishers, The...

D.Y. Ryu et al.

J. Ferment. Technol.

(1972)

C.S. Shin et al.

Biotechnol. Technol.

(1996)

H. Yagi et al.

Ind. Eng. Chem. Proc. Des. Dev.

(1975)

Cited by (100)

Effects of influent physicochemical characteristics on air dissolution, bubble size and rise velocity in dissolved air flotation: A review
2022, Separation and Purification Technology
Microbubble (MB) aided clarification is applied in many industries due to its high efficiency, high loading rate and compact unit size over most other conventional technologies. Compared to coarser air bubbles, MBs perform better in fine particle capture due to a high surface area to volume ratio and increased bubble population. The most common method of generating MBs in water/ wastewater treatment and fine particle recovery in the mineral processing industry is dissolved air flotation (DAF). DAF is a proven efficient technology in water treatment and has been regularly used in this industry over the past few decades. Influent physicochemical parameters can have greater impacts on DAF performance when it is applied in high solid content applications. Air dissolution within the saturator, bubble size distribution and rise velocity of air bubbles are key factors that determine the process efficiency of DAF units. Therefore, understanding the effects of various physicochemical parameters of the influent on DAF performance is important as these variations have to be compensated during both the design and operational stages to obtain better separation efficiencies. The current article reviews up to date research findings on the effect of viscosity, salinity, temperature, pH, zeta potential, surface tension, solid content, particle size and hydraulic loading rate on air dissolution, bubble size distribution and bubble rise velocity of MBs. Finally, conclusions have been drawn from the critical analysis and literature survey and the future research prospects are proposed.
Engineering aspects for scale-up of bioreactors
2022, Current Developments in Biotechnology and Bioengineering: Advances in Bioprocess Engineering
The definition of bioprocesses’ parameters generally occurs at bench scale. However, the reproduction of these processes’ conditions at pilot and/or industrial scale is certainly a challenge. These conditions are for example aeration, which involves critical aspects such as dissolved oxygen and oxygen uptake rate, and agitation, which promotes shear stress and needs high energy consumption. It is important not to lose process efficiency and productivity, which impact on bioeconomy of biotechnological processes. So, some well-known scale-up strategies can be employed, combined or individually, to maintain bioprocesses’ performance. These strategies consider geometric similarity aspects of bioreactors, agitation and aeration conditions, and some rheological aspects of the fluid that must be considered and maintained at the new scale. Some operational conditions have a great influence on cell growth and on the biosynthesis of different biomolecules and should be reproduced at higher scales. Accordingly, one or more operating factors can be maintained constant during scale-up and some procedures can be employed, for example, to determine the power consumption of large-scale bioreactors or determine aeration conditions in an aerobic culture. Stirred tank reactors will be employed as models for scale-up. However, the scale-up of other bioreactors models such as bubble column reactors and solid-state bioreactors will also be described.
Fermentation and downstream processing: Part 1
2022, Integration and Optimization of Unit Operations: Review of Unit Operations from R&D to Production: Impacts of Upstream and Downstream Process Decisions
Fermentation has been used since ancient times to produce foods such as bread, wine, beer, and cheese. While these and other food products are still made by the actions of microorganisms, relatively recent advances in molecular biology and biochemical engineering have allowed the cost-effective application of fermentation to manufacture a wide variety of products over a range of industries. The fermentation scientist and biochemical engineer are tasked with providing the microorganism with the nutrients and environmental conditions that lead to healthy growth, and in turn, economic levels of the desired product. An important part of the challenge is the need to exclude all organisms except the one being cultivated, which requires specific equipment design features and sterilization protocols. For aerobic fermentations, another challenge stems from the exceedingly low solubility of oxygen in water, so that intense mixing is needed to achieve the gas-to-liquid oxygen transfer rate required to sustain the organism. Moreover, aerobic organisms derive energy by oxidizing a substrate such as glucose all the way to carbon dioxide and water, a process that generates a substantial amount of heat. The fermenter designer must provide for removal of this heat, because the organism will not grow if the temperature falls outside of a narrow physiologically compatible range. Lastly, scale-up of aerobic fermentation processes is highly non-linear, and successful translation of laboratory or pilot plant results to production scale requires judicious application of a suitable scale-up method. This chapter explores these and other topics, including microbiology and biochemistry basics, growth kinetics and substrate utilization, and commercial sources of required nutrients. Additional topics are covered in Chapter 3.
Estimation of volumetric mass transfer coefficient (k<inf>L</inf>a)—Review of classical approaches and contribution of a novel methodology
2020, Biochemical Engineering Journal
The volumetric mass transfer coefficient, usually denoted by k_La, is an important parameter for both the design, scale-up and monitoring of aerated bioreactors. Significant research efforts have been expended over the last decades in order to develop reliable and easy to apply methodologies for determining k_La using aeration experiment and fast oxygen (polarographic) probes. These methodologies have been reviewed in this study followed by the presentation of a new and promising methodology for k_La determination, which can be used when data from step experiments are not available. Step and aeration experiments have been carried out in laboratory scale bioreactors and the experimental data were used for the validation of the proposed methodology and also to critically assess all alternative methodologies for estimating k_La.
Enhanced production of anthraquinones by gamma-irradiated cell cultures of Rubia cordifolia in a bioreactor
2020, Industrial Crops and Products
Citation Excerpt :
During bioprocessing, a bioreactor equipped with a Rushton turbine conveys a radial flow to the suspension cultures. Although the proper transfer of oxygen is provided, this setup generates nonuniform mixtures with high shear stress near the blade tips and mild shear at the bioreactor periphery (Badino et al., 2001; Patel et al., 2009). These findings suggested that an appropriate impeller in the bioreactor favored biomass accumulation and secondary metabolite production.
The aim of this study was to obtain high-yielding cell cultures of Rubia cordifolia by applying gamma irradiation and subsequently scaling up for anthraquinone production in a bioreactor. Calli cultured on MS medium was irradiated at variable doses between 2 Gy and 30 Gy. The callus cultures that were irradiated at 8 Gy accumulated a maximum alizarin level of 26.86 mg g^-1 DW and a purpurin level of 44.85 mg g^-1 DW during the M₁T₄ (fourth sub-cultures after gamma irradiation treatment) subculture, which was 6-fold and 11-fold higher than those of the nonirradiated callus cultures, respectively. Suspension cultures that originated from high-yielding callus cultures irradiated at 8 Gy were scaled up in an 8-L stirred bioreactor equipped with different impellers. The callus cultures in a bioreactor using a helical ribbon for homogenous mixing synthesized 63.58 % more anthraquinones than those in the bioreactor equipped with a Rushton disc turbine. This is the first report on the production of alizarin and purpurin in a bioreactor.
Oxygen mass transfer limitations set the performance boundaries of microbial PHA production processes – A model-based problem investigation supporting scale-up studies
2019, Biochemical Engineering Journal
The microbial production of polyhydroxyalkanoates has been reported as a complex process with numerous parameters affecting its performance. Although a great deal of effort has been devoted to the investigation of crucial operating variables, via accurate dynamic polymerization/metabolic/macroscopic models, little work has been implemented on the development of a detailed oxygen mass transfer model. In this study a sophisticated model for PHA producing bacterial cultures is developed, accounting for oxygen mass transfer phenomena in a bioreactor. The volumetric mass transfer coefficient, k_La, is computed as the product of two individual factors, calculated separately with respect to operational conditions, geometrical parameters and culture physiochemical properties. Moreover, an enhancement/prevention factor of oxygen transfer rate (OTR) is taken into consideration. OTR is enhanced due to the oxygen consumption by the cells, and prevented because of the physical blocking effect originated by the cells and excreted biosurfactants, forming thin layers in the gas-liquid interface. Upon integration of the present oxygen mass transfer model to a pre-existed metabolic/macroscopic one, four different operating policies for the progressive improvement of selected process performance indices are tested. Model predictions prove a severe OTR reduction and consequent culture suffocation because of mass transfer limitations in high cell concentrations and biopolymer accumulations regimes. The latter reveals the respective restrictions on the biological process performance and points out the necessity for model-based optimization algorithms.

View all citing articles on Scopus

View full text

Volumetric oxygen transfer coefficients (kLa) in batch cultivations involving non-Newtonian broths

Abstract

Introduction

Section snippets

Microorganism and culture media

Rheological behaviour of fermentation broths

Conclusions

Proc. Biochem.

Biochem. Eng. J.

J. Chem. Tech. Biotechnol.

Biotechnol. Adv.

Acta Biotechnol.

Biotechnol. Bioeng.

The Can. J. Chem. Eng.

Ind. Eng. Chem.

Biotechnol. Bioeng.

J. Ferment. Technol.

Biotechnol. Technol.

Ind. Eng. Chem. Proc. Des. Dev.

Volumetric oxygen transfer coefficients (k_La) in batch cultivations involving non-Newtonian broths