Determination of specific oxygen uptake rate of Photorhabdus luminescens during submerged culture in lab scale bioreactor

Photorhabdus luminescens, a bacterial symbiont of entomoparasitic nematodes, was cultured in a 10 L bioreactor. Cellular density and bioluminescence were recorded and volumetric oxygen transfer coefficient (k_La) and specific oxygen transfer rates were determined during the batch process. Exponential phase of the bacterium lasted for 20 h, showing a maximum specific growth rate of 0.339 h^?1 in a defined medium. Bioluminescence peaked within 21h, and was maintained until the end of the batch process (48 h). The specific oxygen uptake rate (SOUR) was high during both lag and early exponential phase, and eventually reached a stable value of 0.33 mmol g^?1 h^?1 during stationary phase. Maintenance of 200 rpm agitation and 1.4 volume of air per volume of medium per minute (vvm) aeration, gave rise to a k_La value of 39.5 h^?1. This k_La value was sufficient to meet the oxygen demand of 14.4 g L^?1 (DCW) biomass. This research is particularly relevant since there are no reports available on SOURs of symbiotic bacteria or their nematode partners. The insight gained through this study will be useful during the development of a submerged monoxenic culture of Heterorhabditis bacteriophora and its symbiotic bacterium P. luminescens in bioreactors.     

A new dynamic model for highly efficient mass transfer in aerated bioreactors and consequences for kLa identification

Gas–liquid mass transfer is often rate‐limiting in laboratory and industrial cultures of aerobic or autotrophic organisms. The volumetric mass transfer coefficient k_La is a crucial characteristic for comparing, optimizing, and upscaling mass transfer efficiency of bioreactors. Reliable dynamic models and resulting methods for parameter identification are needed for quantitative modeling of microbial growth dynamics. We describe a laboratory‐scale stirred tank reactor (STR) with a highly efficient aeration system (k_La ≈ 570 h^?1). The reactor can sustain yeast culture with high cell density and high oxygen uptake rate, leading to a significant drop in gas concentration from inflow to outflow (by 21%). Standard models fail to predict the observed mass transfer dynamics and to identify k_La correctly. In order to capture the concentration gradient in the gas phase, we refine a standard ordinary differential equation (ODE) model and obtain a system of partial integro‐differential equations (PIDE), for which we derive an approximate analytical solution. Specific reactor configurations, in particular a relatively short bubble residence time, allow a quasi steady‐state approximation of the PIDE system by a simpler ODE model which still accounts for the concentration gradient. Moreover, we perform an appropriate scaling of all variables and parameters. In particular, we introduce the dimensionless overall efficiency κ, which is more informative than k_La since it combines the effects of gas inflow, exchange, and solution. Current standard models of mass transfer in laboratory‐scale aerated STRs neglect the gradient in the gas concentration, which arises from highly efficient bubbling systems and high cellular exchange rates. The resulting error in the identification of κ (and hence k_La) increases dramatically with increasing mass transfer efficiency. Notably, the error differs between cell‐free and culture‐based methods of parameter identification, potentially confounding the determination of the “biological enhancement” of mass transfer. Our new model provides an improved theoretical framework that can be readily applied to aerated bioreactors in research and biotechnology. Biotechnol. Bioeng. 2012; 109: 2997–3006. © 2012 Wiley Periodicals, Inc.     

Design and performance of a new microcalorimetric system for aerobic cultivation of microorganisms

A twin-type heat-conduction calorimeter for an aerobic fermentation process was designed and constructed fermentor both in batch and continuous runs. The time constant of the calorimeter was 3.3 min. The volumetric oxygen transfer coefficient (k_La) of the vessel was measured by a sulfite oxidation method in a continuous flow system under various rotation speeds and gas flow rates. Sufficient thermal stability of the calorimeter was obtained both in batch and continuous runs within the operation time period.     

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.     

Oxygen transfer in an airlift reactor with multiple net draft tubes

A pilot scale airlift reactor with multiple net draft tubes was developed to improve oxygen transfer in the reactor. The reactor was 0.29 m in diameter and 2 m height. A steadystate sulfite oxidation method was applied to determine an overall volumetric mass transfer coefficient. Oxygen transfer of the proposed airlift reactor can be 60–100% higher than that of bubble columns under the same operating conditions.List of Symbols C ^* mol·dm^–3 saturated concentration of dissolved oxygen - C _L mol·dm^–3 bulk concentration of dissolved oxygen - G mol/min nitrogen flow rate - k _L a hr^–1 the volumetric gas-liquid mass transfer coefficient - Mo ₂ g/mol molecular weight of oxygen - OTR g/min the oxygen transfer rate - U _g cm/s superficial air velocity - V _L dm³ volume of the liquid phase - _in oxygen mole ratio in the inlet gas - _out oxygen mole ratio in the outlet gas     

Enhancement factor for oxygen absorption into fermentation broth

Values of the enhancement factor for oxygen absorption into fermentation broth, i.e., the ratio of the liquid phase mass transfer coefficients for oxygen absorption for both cases with and without respiration of microorganisms were predicted theoretically on the assumption of various cell concentration distributions. Calculations indicate that in the usual case the enhancement factor is only slightly or negligibly larger than unity, even when accumulation of microorganisms at or near the gas-liquid interface is assumed. Results of experiments with sparged-stirred fermentors on oxygen absorption into fermentation broths containing resting and growing cells of Candida tropicalis confirmed the theoretical prediction. Except for extreme cases, the effect of respiration of microorganisms on k_La, values can practically be ignored.     

Oxygen mass transfer and scale-up studies in baffled roller bioreactors

Oxygen mass transfer was studied in conventional, bead mill and baffled roller bioreactors. Using central composite rotational design, impacts of size, rotation speed and working volume on the oxygen mass transfer were evaluated. Baffled roller bioreactor outperformed its conventional and bead mill counterparts, with the highest k _L a obtained in these configurations being 0.58, 0.19, 0.41 min^?1, respectively. Performances of the bead mill and baffled roller bioreactor were only comparable when a high bead loading (40 %) was applied. Regardless of configuration increase in rotation speed and decrease in working volume improved the oxygen mass transfer rate. Increase in size led to enhanced mass transfer and higher k _L a in baffled roller bioreactor (0.49 min^?1 for 2.2 L and 1.31 min^?1 for 55 L bioreactors). Finally, the experimentally determined k _L a in the baffled roller bioreactors of different sizes fit reasonably well to an empirical correlation describing the k _L a in terms of dimensionless numbers.     

Determination of volumetric oxygen transfer coefficient by off-gas analysis

In various aerobic bioreactors including activated sludge aeration tanks, the volumetric mass transfer coefficient K_La is frequently used as an estimate of the rate of oxygen dissolution into the liquid phase. The K_La measurement in such bioreactors is widely applied with the aid of sodium sulfite (Na₂SO₃) as an oxygen-consuming substance used to maintain low dissolved oxygen concentration. In the present study, the effect of the addition of Na₂SO₃ on K_La, determined by an off-gas analysis, was investigated specifically from the viewpoint of variations in the size of air bubbles and the enhancement factor associated with the change in sulfite concentration. Experiments were conducted in a draft-tube bubble column, using a zirconia electrode oxygen analyzer for measurement of the O₂ mole fraction in the exhaust gas and a dual electrical resistivity probe for measurement of the bubble size. It was found that the increase in the specific gas-liquid interfacial area, resulting from bubble size reduction effected by Na₂SO₃ functioning as an electrolyte, is more pronounced than the enhancement of the absorption rate through the interface. The upper limit of Na₂SO₃ concentration for sustaining physical absorption, in the absence of any catalyst, ranges from 30 to 70 mol/m³, while that for preventing the average bubble size from decreasing is about 15 mol/m³. Furthermore, to secure a reliable K_La measurement, the K_La value should not exceed 50 h⁻¹ for the liquid depth of 3 m even when the limiting conditions are not exceeded. The off-gas analysis proposed in this study for K_La determination is expected to be extremely useful provided that the above conditions are fulfilled, since it only requires moderate addition of the sulfite as the oxygen-consuming substance and will not interrupt the reactor operation as long as oxygen uptake occurs in the system.     

Effect of n-dodecane on Crypthecodinium cohnii fermentations and DHA production

The potential use of n-dodecane as an oxygen vector for enhancement of Crypthecodinium cohnii growth and docosahexaenoic acid (DHA) production was studied. The volumetric fraction of oxygen vector influenced the gas–liquid volumetric mass transfer coefficient k _L a positively. The k _L a increased almost linearly with the increase of volumetric fraction of n-dodecane up to 1%. The stirring rate showed a higher influence on the k _L a than the aeration rate. The effects of this hydrocarbon on C. cohnii growth and DHA production were then investigated. A control batch fermentation without n-dodecane addition (CF) and a batch fermentation where n-dodecane 1% (v/v) was added (DF) were carried out simultaneously under the same experimental conditions. It was found that, before 86.7 h of fermentation, the biomass concentration, the specific growth rate, the DHA, and total fatty acids (TFA) production were higher in the CF. After this fermentation time, the biomass concentration, the DHA and TFA production were higher in the DF. The highest DHA content of biomass (6.14%), DHA percentage of TFA (51%), and DHA production volumetric rate r _DHA (9.75 mg l⁻¹ h⁻¹) were obtained at the end of the fermentation with n-dodecane (135.2 h). The dissolved oxygen tension (DOT) was always higher in the DF, indicating a better oxygen transfer due to the oxygen vector presence. However, since the other C. cohnii unsaturated fatty acids percentages did not increase with the oxygen availability increase due to the n-dodecane presence, a desaturase oxygen-dependent mechanism involved in the C. cohnii DHA biosynthesis was not considered to explain the DHA production increase. A selective extraction through the n-dodecane was suggested.     

Microcalorimetric investigations of the metabolism of yeasts. growth in batch and chemostat cultures on ethanol medium

Summary In the presence of protein, Hansenula polymorpha cultivation medium exhibits a maximum volumetric mass transfer coefficient, k_La, as function of the employed antifoam agents (soy oil and Desmophen 3600). With diminishing superficial gas velocity this maximum disappeas.Symbols E_G Relative gas holdup - k_La Volumetric mass transfer coefficient (s^–1) - w_SL Superficial liquid velocity (cm s^–1) - w_SG Superficial gas velocity (cm s^–1)     

Scale-up and application of a cyclone reactor for fermentation processes

A cyclone reactor for microbial fermentation processes was developed with high oxygen transfer capabilities. Three geometrically similar cyclone reactors with 0.5?l, 2.5?l and 15?l liquid volume, respectively, were characterized with respect to oxygen mass transfer, mixing time and residence time distribution. Semi-empirically correlations for prediction of oxygen mass transfer and mixing times were identified for scale-up of cyclone reactors. A volumetric oxygen mass transfer coefficient k _L a of 1.0?s^?1 (available oxygen transfer rate with air: 29?kg?m^?3?h^?1) was achieved with the cyclone reactor at a volumetric power input of 40?kW?m^?3 and an aeration gas flow rate of 0.2?s^?1. Continuous methanol controlled production of formate dehydrogenase (FDH) with Candida boidinii in a 15?l cyclone reactor resulted in more than 100% improvement in dry cell mass concentration (64.5?g?l^?1) and in about 100% improvement in FDH space-time yield (300?U?l^?1?h^?1) compared to steady state results of a continuous stirred tank reactor.     

Optimization of lipase production by <Emphasis Type="Italic">Staphylococcus warneri</Emphasis> EX17 using the polydimethylsiloxanes artificial oxygen carriers

In this research, the combined effects of polydimethylsiloxane (PDMS) and different conditions of oxygen volumetric mass transfer coefficient (k_La) on lipase production by Staphylococcus warneri EX17 were studied and optimized in bioreactor cultures. Raw glycerol from biodiesel synthesis was used as the sole carbon source. Full-factorial central composite design and the response surface methodology were employed for the experimental design and analysis of the results. The optimal polydimethylsiloxane concentration and mass coefficient transfer (k_La) were found to be 13.5% (v/v) and 181 h⁻¹, respectively. Under these conditions, the maximal cell production obtained was 10.0 g/l, and the volumetric lipase activities of approximately 490 U/l, after 6 h of cultivation. These results are in close agreement with the model predictions. Results obtained in this work reveal the positive effects of PDMS on oxygen volumetric mass transfer coefficient (k_La) in the Staphylococcus warneri EX17 cultivation and lipase production.     

Effects of hydrocarbon additions on gas-liquid mass transfer coefficients in biphasic bioreactors

The effects of aliphatic hydrocarbons (n-hexadecane andn-dodecane) on the volumetric oxygen mass transfer coefficient (k _L a) were studied in flat alveolar airlift reactor and continuous stirred tank reactors (CSTRs). In the flat alveolar airlift reactor, high aeration rates (>2 vvm) were required in order to obtain efficient organic-aqueous phase dispersion and reliablek _L a measurements. Addition of 1% (v/v)n-hexadecane orn-dodecane increased thek _l a 1.55-and 1.33-fold, respectively, compared to the control (superficial velocity: 25.8×10⁻³ m/s, sparger orifice diameter: 0.5 mm). Analysis of the gas-liquid interfacial areaa and the liquid film mass transfer coefficientk _L suggests that the observedk _L a increase was a function of the media's liquid film mass transfer. Addition of 1% (v/v)n-hexadecane orn-dodecane to analogous setups using CSTRs led to ak _L a increase by a factor of 1.68 and 1.36, respectively (superficial velocity: 2.1×10⁻³ m/s, stirring rate: 250 rpm). These results propose that low-concentration addition of oxygen-vectors to aerobic microbial cultures has additional benefit relative to incubation in purely aqueous media.     

Bubble coalescence behaviour in biological media

Summary A twin bubble column was used to measure the k_La values for oxygen in model and cultivation media using the steady state method described previously (Adler et al. 1980). Desmophen and soy oil were used as antifoam agents together with model and/or cultivation media for Chaetomium cellulotyticum, Trichoderma reesei, Hansenula polymorpha, Saccharomyces cerevisiae and Escherichia coli. The bubble coalescence behavior is mainly influenced by antifoam agents and somewhat by protein and alcohol additives. In the range investigated (0.01 to 0.1%.), the k_La values are not influenced by the Desmophen concentration and only slighthly by the soy oil concentration (0.5 to 1.5%.). The coalescence behaviour was characterized by the ratio m_corr=(k_La)_corr/(k_La)_ref. A nutrient salt solution with Desmophen was used as a reference. The k_La measured in the investigated media were corrected by considering the differences in k_La's in the investigated and reference media. These m_corr values can directly be used for bubble columns close to the optimum aeration rate.Symbols a Specific gas/liquid interfacial area - c Concentration - k_L Mass transfer coefficient - k_La Volumetric mass transfer coefficient - W_SG Superficial gas velocity - E_G Relative gas hold-up     

Studies on fluid dynamics of the flow field and gas transfer in orbitally shaken tubes

Orbitally shaken cylindrical bioreactors [OrbShake bioreactors (OSRs)] without an impeller or sparger are increasingly being used for the suspension cultivation of mammalian cells. Among small volume OSRs, 50‐mL tubes with a ventilated cap (OSR50), originally derived from standard laboratory centrifuge tubes with a conical bottom, have found many applications including high‐throughput screening for the optimization of cell cultivation conditions. To better understand the fluid dynamics and gas transfer rates at the liquid surface in OSR50, we established a three‐dimensional simulation model of the unsteady liquid forms (waves) in this vessel. The studies verified that the operating conditions have a large effect on the interfacial surface. The volumetric mass transfer coefficient (k_La) was determined experimentally and from simulations under various working conditions. We also determined the liquid‐phase mass transfer coefficient (k_L) and the specific interfacial area (a) under different conditions to demonstrate that the value of a affected the gas transfer rate more than did the value of k_L. High oxygen transfer rates, sufficient for supporting the high‐density culture of mammalian cells, were found. Finally, the average axial velocity of the liquid was identified to be an important parameter for maintaining cells in suspension. Overall these studies provide valuable insights into the preferable operating conditions for the OSR50, such as those needed for cell cultures requiring high oxygen levels. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:192–200, 2017     

Oxygen absorption in stirred tanks: A correlation for ionic strength effects

A new correlation is given for the prediction of the volumetric coefficient for mass transfer (K_La) in stirred tanks from dispersed gas bubbles to basal salt solutions of ionic strengths representative of fermentation media. The correlation includes the effects of both the operating parameters (agitation power per unit volume and gas superficial velocity) and the physicochemical properties of the system: interfacial tension, viscosity, density, diffusion, coefficient and, in particular, ionic strength. The effect of the latter was found to be most significant in the Newtonian systems of water-like viscosity investigated; no previous correlations have included the effect of ionic strength. K_La values were determined by using a dissolved oxygen probe to monitor the steady-state oxygen tension in continuous flow experiments, and/or the rate of change of oxygen tension in unsteady-state semibatch experiments. In the latter cases, results were computed by a nonlinear, least squares computer program which fitted the experimental data to a model of probe transient response characteristics. The general applicability of the model and the computational procedure was verified by comparing the results to those obtained with the same electrolyte solution in the steady-state mode. The experiments were run over a wide range of agitation power inputs, including those typical of both soluble- and insoluble-substrate fermentations. The correlation appears to be valid for both oxygen mass transfer with and without homogeneous chemical reaction in the liquid phase; in the former case, for example, sulfite oxidation, knowledge of the chemical reaction enhancement factor is required. In addition to predicting oxygen transfer capabilities, the correlation may be used for other sparingly soluble gases of interest in fermentation systems, such as methane, hydrogen, and carbon dioxide.     

Syngas fermentation to biofuel: Evaluation of carbon monoxide mass transfer coefficient (kLa) in different reactor configurations

Lignocellulosic biomass such as agri‐residues, agri‐processing by‐products, and energy crops do not compete with food and feed, and is considered to be the ideal renewable feedstocks for biofuel production. Gasification of biomass produces synthesis gas (syngas), a mixture primarily consisting of CO and H₂. The produced syngas can be converted to ethanol by anaerobic microbial catalysts especially acetogenic bacteria such as various clostridia species.One of the major drawbacks associated with syngas fermentation is the mass transfer limitation of these sparingly soluble gases in the aqueous phase. One way of addressing this issue is the improvement in reactor design to achieve a higher volumetric mass transfer coefficient (k_La). In this study, different reactor configurations such as a column diffuser, a 20‐μm bulb diffuser, gas sparger, gas sparger with mechanical mixing, air‐lift reactor combined with a 20‐μm bulb diffuser, air‐lift reactor combined with a single gas entry point, and a submerged composite hollow fiber membrane (CHFM) module were employed to examine the k_La values. The k_La values reported in this study ranged from 0.4 to 91.08 h^?1. The highest k_La of 91.08 h^?1 was obtained in the air‐lift reactor combined with a 20‐μm bulb diffuser, whereas the reactor with the CHFM showed the lowest k_La of 0.4 h^?1. By considering both the k_La value and the statistical significance of each configuration, the air‐lift reactor combined with a 20‐μm bulb diffuser was found to be the ideal reactor configuration for carbon monoxide mass transfer in an aqueous phase. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Bubble coalescence behaviour in biological media

Summary Volumetric mass transfer coefficients (k_La) were measured by a steady state method in a twin bubble column to characterize the coalescence behaviour of the medium. Employing Hansenula polymorpha cultivation broths, k_La values were compared with those measured in model media in the presence and absence of antifoam agents. The ratio of the volumetric mass transfer coefficient in the system investigated to that in water, , was employed to characterize the cultivation medium.Symbols a Specific gas/liquid interfacial area with regard to the liquid volume in reactor - d_e Dynamical equilibrium bubble diameter - d_H Perforated plate hole diameter - d_p Primary bubble diameter - d_S Sauter bubble diameter - F_v Liquid feed rate - H Bubbling layer height - k_L Gas/liquid mass transfer coefficient - k_La Volumetric mass transfer coefficient - m k_La/(k_La)_r coalescence index - m_corr Corrected coalescence index [Eq. (3)] - OTR Oxygen transfer rate - P_O Dissolved O₂-partial pressure in BS2 - P₁ Dissolved O₂-partial pressure in BS1 - P_O P_O/P_S relative oxygen saturation in BS2 - P₁ P₁/P_S relative oxygen saturation in BS1 - P_S Saturation dissolved oxygen partial pressure - R_c dn_B/dt coalescence rate - S Substrate concentration - t_F Time since the beginning of the cultivation - X Biomass concentration - V₁ Liquid volume in BS1 - w_SG Superficial gas velocity in BS1 - G Gas holdup in BS1 - ₁ V₁/F_v mean liquid residence time in BS1 - BS1 O₂ absorber column - BS2 O₂ desorber column - D Desmophen (antifoam agent) - NS Nutrient salt solution (Table 1)     

Gas–liquid mass transfer in an up-flow cocurrent packed-bed biofilm reactor

Gas–liquid mass transfer was investigated in an up-flow cocurrent packed-bed biofilm reactor. In aerobic processes gas–liquid mass transfer can be considered as a key operational parameter as well as in reactor scale-up. The present paper investigates the influence of the liquid phase mixing in the determination of the volumetric gas–liquid mass transfer coefficient (k_La) coefficient. Residence time distribution (RTD) experiments were performed in the reactor to determine the flow pattern of the liquid phase and to model mathematically the liquid phase mixing. The mathematical model derived from RTD experiments was used to evaluate the influence of the liquid mixing on the experimental estimation of the (k_La) in this reactor type. The methods used to estimate the k_La coefficient were: (i) dynamic gassing-out, (ii) sulphite method, and (iii) in-process estimation through biological conversion obtained in the reactor. The use of standard chemical engineering correlations to determine the k_La in this type of bioreactors is assessed. Experimental and modelling results show how relevant can be to take into consideration the liquid phase mixing in the calculations of the most-used methods for the estimation of k_La coefficient. k_La coefficient was found to be strongly heterogeneous along the reactor vertical axis. The value of the k_La coefficient for the packed-bed section ranged 0.01–0.12 s⁻¹. A preliminary correlation was established for up-flow cocurrent packed-bed biofilm reactors as a function of gas superficial velocity.     

Influence of oxygen transfer rate on the accumulation of poly(3-hydroxybutyrate) by Bacillus megaterium

Poly(3-hydroxybutyrate)—P(3HB)—is a natural biodegradable polyester synthesized by several bacteria, produced from renewable resources. The effects of oxygen transfer rate on the intracellular accumulation of P(3HB) was evaluated, aiming at increasing P(3HB) synthesized by Bacillus megaterium DSM 32^T in bioreactor batch cultures. Bench-scale bioreactor cultivations were performed under different volumetric oxygen mass transfer coefficients, k_La, setting stirrer speed on specified values. The results of this work show that oxygen transfer is a key factor on P(3HB) accumulation by B. megaterium, increasing the P(3HB) intracellular mass fraction from 39% to 62% of CDW at k_La condition of 0.006 s^?1.