Rapid depletion of dissolved oxygen in 96‐well microtiter plate Staphylococcus epidermidis biofilm assays promotes biofilm development and is influenced by inoculum cell concentration

Biofilm‐related research using 96‐well microtiter plates involves static incubation of plates indiscriminate of environmental conditions, making oxygen availability an important variable which has not been considered to date. By directly measuring dissolved oxygen concentration over time we report here that dissolved oxygen is rapidly consumed in Staphylococcus epidermidis biofilm cultures grown in 96‐well plates irrespective of the oxygen concentration in the gaseous environment in which the plates are incubated. These data indicate that depletion of dissolved oxygen during growth of bacterial biofilm cultures in 96‐well plates may significantly influence biofilm production. Furthermore higher inoculum cell concentrations are associated with more rapid consumption of dissolved oxygen and higher levels of S. epidermidis biofilm production. Our data reveal that oxygen depletion during bacterial growth in 96‐well plates may significantly influence biofilm production and should be considered in the interpretation of experimental data using this biofilm model. Biotechnol. Bioeng. 2009;103: 1042–1047. © 2009 Wiley Periodicals, Inc.     

Balancing redox cofactor generation and ATP synthesis: Key microaerobic responses in thermophilic fermentations

Geobacillus thermoglucosidasius is a Gram‐positive, thermophilic bacterium capable of ethanologenic fermentation of both C5 and C6 sugars and may have possible use for commercial bioethanol production [Tang et al., 2009; Taylor et al. (2009) Trends Biotechnol 27(7): 398–405]. Little is known about the physiological changes that accompany a switch from aerobic (high redox) to microaerobic/fermentative (low redox) conditions in thermophilic organisms. The changes in the central metabolic pathways in response to a switch in redox potential were analyzed using quantitative real‐time PCR and proteomics. During low redox (fermentative) states, results indicated that glycolysis was uniformly up‐regulated, the Krebs (tricarboxylic acid or TCA) cycle non‐uniformly down‐regulated and that there was little to no change in the pentose phosphate pathway. Acetate accumulation was accounted for by strong down‐regulation of the acetate CoA ligase gene (acs) in addition to up‐regulation of the pta and ackA genes (involved in acetate production), thus conserving ATP while reducing flux through the TCA cycle. Substitution of an NADH dehydrogenase (down‐regulated) by an up‐regulated NADH:FAD oxidoreductase and up‐regulation of an ATP synthase subunit, alongside the observed shifts in the TCA cycle, suggested that an oxygen‐scavenging electron transport chain likely remained active during low redox conditions. Together with the observed up‐regulation of a glyoxalase and down‐regulation of superoxide dismutase, thought to provide protection against the accumulation of toxic phosphorylated glycolytic intermediates and reactive oxygen species, respectively, the changes observed in G. thermoglucosidasius NCIMB 11955 under conditions of aerobic‐to‐microaerobic switching were consistent with responses to low pO₂ stress. Biotechnol. Bioeng. 2013; 110: 1057–1065. © 2012 Wiley Periodicals, Inc.     

Morphology engineering of Aspergillus niger for improved enzyme production

Supplementation with silicate microparticles was used as novel approach to control the morphological development of Aspergillus niger, important as the major world source of citric acid and higher‐value enzymes, in submerged culture. With careful variation of size and concentration of the micromaterial added, a number of distinct morphological forms including pellets of different size, free dispersed mycelium, and short hyphae fragments could be reproducibly created. Aluminum oxide particles similarly affected morphology, showing that this effect is largely independent of the chemical particle composition. Image analysis of morphological development of A. niger during the cultivation process showed that the microparticles influence the morphology by collision‐induced disruption of conidia aggregates and probably also the hindrance of new spore–spore interactions in the very early stage of the process. Exemplified for different recombinant A. niger strains enzyme production could be strongly enhanced by the addition of microparticles. Linked to the formation of freely dispersed mycelium, titers for glucoamylase (GA) expressed as intracellular enzyme (88 U/mL) and fructofuranosidase secreted into the supernatant (77 U/mL), were up to fourfold higher in shake flasks. Moreover, accumulation of the undesired by‐product oxalate was suppressed by up to 90%. The microparticle strategy could be successfully transferred to fructofuranosidase production in bioreactor, where a final titer of 160 U/mL could be reached. Using co‐expression of GA with green fluorescent protein, enzyme production was localized in the cellular aggregates of A. niger. For pelleted growth, protein production was maximal only within a thin layer at the pellet surface and markedly decreased in the pellet interior, whereas the interaction with the microparticles created a highly active biocatalyst with the dominant fraction of cells contributing to production. Biotechnol. Bioeng. 2010;105: 1058–1068. © 2009 Wiley Periodicals, Inc.     

Compatible solutes of sclerotia of Mycoleptodiscus terrestris under different culture and drying conditions

Mycoleptodiscus terrestris is a plant pathogen which has been shown to be effective in controlling invasive aquatic weeds in inundative biocontrol applications. The preferred propagule for production and application is the sclerotium. In the current study, we evaluated the accumulation of carbohydrates and polyols in the sclerotia of M. terrestris under different culture and drying regimes. The carbohydrates and polyols screened for represent a class of compatible solutes found in fungi. The results show mannitol and trehalose are the major analytes identified in the sclerotia of M. terrestris. We identified higher levels of mannitol and trehalose in liquid-culture produced samples relative to solid-state produced samples. The impact of fermentation time was examined and shown to impact solute levels. In addition, the drying regime was varied to produce samples dried to different moisture contents. The experiments show greater drying led to higher mannitol levels, while trehalose levels remained constant. Rapid drying of the sclerotia in a fluidized-bed dryer also show rapid accumulation of mannitol and trehalose, which suggest the enzyme activities needed for production are readily available. The findings confirm mannitol and trehalose are important metabolites in M. terrestris and their concentrations are responsive to osmotic stress conditions.     

A genetic and metabolic approach to redirection of biochemical pathways of Clostridium butyricum for enhancing hydrogen production

Clostridium butyricum, a well known H₂ producing bacterium, produces lactate, butyrate, acetate, ethanol, and CO₂ as its main by‐products from glucose. The conversion of pyruvate to lactate, butyrate and ethanol involves oxidation of NADH. It was hypothesized that the NADH could be increased if the formation of these by‐products could be eliminated, resulting in enhancing H₂ yield. Herein, this study aimed to establish a genetic and metabolic approach for enhancing H₂ yield via redirection of metabolic pathways of a C. butyricum strain. The ethanol formation pathway was blocked by disruption of aad (encoding aldehyde‐alcohol dehydrogenase) using a ClosTron plasmid. Although elimination of ethanol formation alone did not increase hydrogen production, the resulting aad‐deficient mutant showed approximately 20% enhanced performance in hydrogen production with the addition of sodium acetate. This work demonstrated the possibility of improving hydrogen yield by eliminating the unfavorable by‐products ethanol and lactate. Biotechnol. Bioeng. 2013; 110: 338–342. © 2012 Wiley Periodicals, Inc.     

Improvement of red pigment/citrinin production ratio as a function of environmental conditions by monascus ruber

The growth and metabolic behaviour of the filamentous fungus Monascus ruber were studied in submerged cultures under various aeration and agitation conditions. Improving the oxygen supply, by increasing either the air input or the agitation speed, resulted in modified metabolism: the biomass yield, the consumption of the nitrogen source (monosodium glutamate), and the production of secondary metabolites (red pigment and citrinin) all increased. However, the citrinin production increased more than that of the red pigment. In consequence, a low oxygen transfer coefficient was required to improve the red pigment/citrinin production ratio. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 497–501, 1999.     

The role of the octadecanoid pathway in the production of terpenoid indole alkaloids in Catharanthus roseus hairy roots under normal and UV‐B stress conditions

The octadecanoid pathway is responsible for producing jasmonic acid an important signaling molecule in plants, which controls the production of a variety of secondary metabolites. Previously the exogenous addition of jasmonic acid to Catharanthus roseus hairy roots caused an increase in terpenoid indole alkaloid (TIA) accumulation. The role of the endogenous production of jasmonic acid by the octadecanoid pathway in the production of TIAs in C. roseus hairy roots is examined. Feeding of octadecanoid pathway inhibitors suggests that the octadecanoid pathway does not actively control TIA production under normal growth conditions or during the UV‐B stress response in C. roseus hairy roots. Biotechnol. Bioeng. 2009;103: 1248–1254. © 2009 Wiley Periodicals, Inc.     

Life‐cycle kinetic model for endospore‐forming bacteria,including germination and sporulation

We develop a mechanistic life‐cycle model for endospore‐forming bacteria (EFB) and test the model with experiments with a Bacillus mixed culture. The model integrates and quantifies how sporulation and germination are triggered by depletion or presence of a limiting substrate, while both substrates affect the rate of vegetative growth by a multiplicative model. Kinetic experiments show the accumulation of small spherical spores after the triggering substrate is depleted, substantially more rapid decay during sporulation than for normal decay of vegetative cells, and a higher specific substrate utilization rate for the germinating cells than that for growth of vegetative cells. Model simulations capture all of these experimental trends. According to model predictions, when a batch reactor is started, seeding with EFB spores instead of active EFB delays the onset of rapid chemical oxygen demand (COD) utilization and biomass growth, but the end points are the same. Simulated results with low aeration intensity show that germination can consume some substrate without dissolved oxygen (DO) depletion. Biotechnol. Bioeng. 2009; 104: 1012–1024. © 2009 Wiley Periodicals, Inc.     

Butyrate production enhancement by Clostridium tyrobutyricum using electron mediators and a cathodic electron donor

Electron mediators and electron supply through a cathode were examined to enhance the reducing power for butyrate production by an acidogenic clostridium strain, Clostridium tyrobutyricum BAS 7. Among the tested electron mediators, methyl viologen (MV)‐amended cultures showed an increase of butyrate productivity (1.3 times), final concentration (1.4 times), and yield (1.3 times). The electron flow altered by MV addition from the ferredoxin pool to the NADH pool was shown by one electron model, implying that more available NADH increased butyrate production. In the cathode compartment poised at ?400 mV versus the Ag/AgCl electrode, the neutral red (NR)‐amended cultures of Clostridium tyrobutyricum BAS 7 increased butyrate concentration (from 5 to 8.8 g/L) and yield (from 0.33 up to 0.44 g/g) with no acetate production at all. Given that electrically reduced NR (NR_red, yellow) by the cathode was re‐oxidized (NR_ox, red) in the cells on the basis of color change, electron flow from NR_red to NAD⁺ (i.e., NADH generation) induced an increase in butyrate production. This is the first report to show the increase of butyric acid production by electrically driven acidogenesis. These results show that the electron flow altered NADH formation by electron mediators and by the cathodic electron donor, increasing the yield and selectivity of reduced end‐products like butyrate. Biotechnol. Bioeng. 2012; 109: 2494–2502. © 2012 Wiley Periodicals, Inc.     

Glycerol production by two filamentous fungi grown at different ionic and nonionic osmotics and cheese whey

Glycerol production by a highly glycerol-producing local isolate (Eurotium amstelodami) and a standard reference isolate (Aspergillus wentii) was markedly enhanced by high saline media. Glycerol concentration depended on the external osmotic. Thus, the highest glycerol concentration was found in the presence of NaCl, followed by KCl, with considerably lower values for MgCl₂ and CaCl₂ saline media. With glucose (5–50%) used as a nonionic osmotic, low levels of glycerol were obtained and the main pool of polyols was mannitol. Glycerol production was gradually increased with the increase of NaCl concentration of cheese whey, reaching maxima by both organisms when whey was supplemented with 8% NaCl (total of 16% NaCl). The quantity of glycerol produced byA. wentii was twice higher than that obtained byE. amstelodami on whey treated with 8% NaCl.     

Optimization of elastin‐like polypeptide fusions for expression and purification of recombinant proteins in plants

The demand for recombinant proteins for medical and industrial use is expanding rapidly and plants are now recognized as an efficient, inexpensive means of production. Although the accumulation of recombinant proteins in transgenic plants can be low, we have previously demonstrated that fusions with an elastin‐like polypeptide (ELP) tag can significantly enhance the production yield of a range of different recombinant proteins in plant leaves. ELPs are biopolymers with a repeating pentapeptide sequence (VGVPG)_n that are valuable for bioseparation, acting as thermally responsive tags for the non‐chromatographic purification of recombinant proteins. To determine the optimal ELP size for the accumulation of recombinant proteins and their subsequent purification, various ELP tags were fused to green fluorescent protein, interleukin‐10, erythropoietin and a single chain antibody fragment and then transiently expressed in tobacco leaves. Our results indicated that ELP tags with 30 pentapeptide repeats provided the best compromise between the positive effects of small ELP tags (n = 5–40) on recombinant protein accumulation and the beneficial effects of larger ELP tags (n = 80–160) on recombinant protein recovery during inverse transition cycling (ITC) purification. In addition, the C‐terminal orientation of ELP fusion tags produced higher levels of target proteins, relative to N‐terminal ELP fusions. Importantly, the ELP tags had no adverse effect on the receptor binding affinity of erythropoietin, demonstrating the inert nature of these tags. The use of ELP fusion tags provides an approach for enhancing the production of recombinant proteins in plants, while simultaneously assisting in their purification. Biotechnol. Bioeng. 2009;103: 562–573. © 2009 Wiley Periodicals, Inc.     

Metabolic engineering of the anaerobic central metabolic pathway in Escherichia coli for the simultaneous anaerobic production of isoamyl acetate and succinic acid

An in vivo method of producing isoamyl acetate and succinate simultaneously has been developed in Escherichia coli to maximize yields of both high value compounds as well as maintain the proper redox balance between NADH and NAD⁺. Previous attempts at producing the ester isoamyl acetate anaerobically did not produce the compound in high concentrations because of competing pathways and the need for NAD⁺ regeneration. The objective of this study is to produce succinate as an example of a reduced coproduct to balance the ratio of NADH/NAD⁺ as a way of maximizing isoamyl acetate production. Because the volatility of the two compounds differs greatly, the two could be easily separated in an industrial setting. An ldhA, adhE double mutant strain (SBS110MG) served as the control strain to test the effect of an additional ackA‐pta mutation as found in SBS990MG. Both strains overexpressed the two heterologous genes pyruvate carboxylase and alcohol acetyltransferase (for ester production). The triple mutant SBS990MG was found to produce higher levels of both isoamyl acetate and succinate. At the optimal condition of 25°C, the culture produced 9.4 mM isoamyl acetate and 45.5 mM succinate. SBS990MG produced 36% more ester and over 700% more succinate than SBS110MG. In addition, this study demonstrated that a significantly higher isoamyl acetate concentration can be attained by simultaneously balancing the carbon and cofactor flow; the isoamyl acetate concentration of 9.4 mM is more than seven times higher than an earlier report of about 1.2 mM. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Development of thermodynamic optimum searching (TOS) to improve the prediction accuracy of flux balance analysis

Flux balance analysis (FBA) has been widely used in calculating steady‐state flux distributions that provide important information for metabolic engineering. Several thermodynamics‐based methods, for example, quantitative assignment of reaction directionality and energy balance analysis have been developed to improve the prediction accuracy of FBA. However, these methods can only generate a thermodynamically feasible range, rather than the most thermodynamically favorable solution. We therefore developed a novel optimization method termed as thermodynamic optimum searching (TOS) to calculate the thermodynamically optimal solution, based on the second law of thermodynamics, the minimum magnitude of the Gibbs free energy change and the maximum entropy production principle (MEPP). Then, TOS was applied to five physiological conditions of Escherichia coli to evaluate its effectiveness. The resulting prediction accuracy was found significantly improved (10.7–48.5%) by comparing with the ¹³C‐fluxome data, indicating that TOS can be considered an advanced calculation and prediction tool in metabolic engineering. Biotechnol. Bioeng. 2013; 110: 914–923. © 2012 Wiley Periodicals, Inc.     

Metabolomics for biotransformations: Intracellular redox cofactor analysis and enzyme kinetics offer insight into whole cell processes

For redox reactions catalyzed by microbial cells the analysis of involved cofactors is of special interest since the availability of cofactors such as NADH or NADPH is often limiting and crucial for the biotransformation efficiency. The measurement of these cofactors has usually been carried out using spectrophotometric cycling assays. Today LC‐MS/MS methods have become a valuable tool for the identification and quantification of intracellular metabolites. This technology has been adapted to measure all four nicotinamide cofactors (NAD, NADP, NADH, and NADPH) during a whole cell biotransformation process catalyzed by recombinant Escherichia coli cells. The cells overexpressing an alcohol dehydrogenase from Lactobacillus brevis were used for the reduction of methyl acetoacetate (MAA) with substrate‐coupled cofactor regeneration by oxidation of 2‐propanol. To test the reliability of the measurement the data were evaluated using a process model. This model was derived using the measured concentrations of reactants and cofactors for initiation as well as the kinetic constants from in vitro measurements of the isolated enzyme. This model proves to be highly effective in the process development for a whole cell redox biotransformation in predicting both the right concentrations of cofactors and reactants in a batch and in a CSTR process as well as the right in vivo expression level of the enzyme. Moreover, a sensitivity analysis identifies the cofactor regeneration reaction as the limiting step in case for the reduction of MAA to the corresponding product (R)‐methyl 3‐hydroxybutyrate. Using the combination of in vitro enzyme kinetic measurements, measurements of cofactors and reactants and an adequate model initiated by intracellular concentrations of all involved reactants and cofactors the whole cell biotransformation process can be understood quantitatively. Biotechnol. Bioeng. 2009; 104: 251–260 © 2009 Wiley Periodicals, Inc.     

Effects of osmotic and matric potential on radial growth and accumulation of endogenous reserves in three isolates of Pochonia chlamydosporia

For the first time, the effects of varying osmotic and matric potential on fungal radial growth and accumulation of polyols were studied in three isolates of Pochonia chlamydosporia. Fungal radial growth was measured on potato dextrose agar modified osmotically using potassium chloride or glycerol. PEG 8000 was used to modify matric potential. When plotted, the radii of the colonies were found to grow linearly with time, and regression was applied to estimate the radial growth rate (mm day^?1). Samples of fresh mycelia from 25-day-old cultures were collected and the quantity (mg g^?1 fresh biomass) of four polyols (glycerol, erythritol, arabitol and mannitol) and one sugar (glucose) was determined using HPLC. Results revealed that fungal radial growth rates decreased with increased osmotic or matric stress. Statistically significant differences in radial growth were found between isolates in response to matric stress (P<0.006) but not in response to osmotic stress (P=0.759). Similarly, differences in the total amounts of polyols accumulated by the fungus were found between isolates in response to matric stress (P<0.001), but not in response to osmotic stress (P=0.952). Under water stress, the fungus accumulated a combination of different polyols important in osmoregulation, which depended on the solute used to generate the stress. Arabitol and glycerol were the main polyols accumulated in osmotically modified media, whereas erythritol was the main polyol that was accumulated in media amended with PEG. The results found that Pochonia chlamydosporia may use different osmoregulation mechanisms to overcome osmotic and matric stresses.     

Scale‐up of citric acid fermentation by redox potential control

To obtain high citric acid productivity in Aspergillus niger fermentation on beet molasses substrate, a certain redox potential profile with two maxima (260 and 280 mV) and two minima (180 and 80 mV) must be maintained. The most effective regulation of redox potential is by regulation of aeration and agitation. It has been shown that control of redox potential by aeration and agitation is a most successful method for scale‐up from 10‐L laboratory scale to the 100‐ and 1000‐L pilot‐plant scale, even in geometrically dissimilar stirred‐tank reactors. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 552–557, 1999.     

Enhancement of seed germination in high salinity by engineering mannitol expression in Arabidopsis thaliana

The bacterial gene mtlD, which encodes mannitol 1-phosphate dehydrogenase (E. C. 1. 1. 1. 17), was transformed into Arabidopsis thaliana and expressed under control of the CaMV 35S promoter. MtlD-transformants accumulated mannitol, a sugar alcohol that is not normally found in Arabidopsis. Amounts of soluble carbohydrates, sucrose, glucose, fructose, myo-inositol and mannitol were determined in different tissues of wild-type and transgenic plants. We estimated that less than 1& of the carbon assimilated was converted into mannitol by the transgenic plants. The establishment of individual transformed lines (after self-crossing three times) resulted in high and low mannitol-producing lines which were stably maintained. The presence of mannitol did not alter plant appearance or growth habit. When MtlD-expressing seeds and control seeds (T3 generation) were imbibed with solutions containing NaCl (range 0 to 400 mol m^?3), transgenic seeds containing mannitol germinated in medium supplemented with up to 400 mol m^?3 NaCl, while control seeds ceased germination at 100 mol m^?3 NaCl. It is doubtful whether the ability to germinate in high salt was a result of an osmotic effect exerted by elevated levels of mannitol, considering that mannitol concentrations were in the mol m^?3 range in seeds. A specific effect of polyols, for example on the integrity of subcellular membranes or enzymes, cannot be excluded.     

Elucidating mechanisms of solvent toxicity in ethanologenic Escherichia coli

Ethanol toxicity and its effect on ethanol production by the recombinant ethanologenic Escherichia coli strain KO11 were investigated in batch and continuous fermentation. During batch growth, ethanol produced by KO11 reduced both the specific cell growth rate (µ) and the cell yield (Y_X/S). The extent of inhibition increased with the production of both acetate and lactate. Subsequent accumulation of these metabolites and ethanol resulted in cessation of cell growth, redirection of metabolism to reduce ethanol production, and increased requirements for cell maintenance. These effects were found to depend on both the glycolytic flux and the flux from pyruvate to ethanol. Pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) activities measured during the batch fermentation suggested that decreased ethanol production resulted from enzyme inhibition rather than down‐regulation of genes in the ethanol‐producing pathway. Ethanol was added in continuous fermentation to provide an ethanol concentration of either 17 or 27 g/L, triggering sustained oscillations in the cell growth rate. Cell concentrations oscillated in‐phase with ethanol and acetate concentrations. The amplitude of oscillations depended on the concentration of ethanol in the fermentor. Through multiple oscillatory cycles, the yield (Y_P/S) and concentration of ethanol decreased, while production of acetate increased. These results suggest that KO11 favorably adapted to improve growth by synthesizing more ATP though acetate production, and recycling NADH by producing more lactate and less ethanol. Implications of these results for strategies to improve ethanol production are described. Biotechnol. Bioeng. 2010;106: 721–730. © 2010 Wiley Periodicals, Inc.     

Mechanistic modeling of sulfur‐deprived photosynthesis and hydrogen production in suspensions of Chlamydomonas reinhardtii

The ability of unicellular green algal species such as Chlamydomonas reinhardtii to produce hydrogen gas via iron‐hydrogenase is well known. However, the oxygen‐sensitive hydrogenase is closely linked to the photosynthetic chain in such a way that hydrogen and oxygen production need to be separated temporally for sustained photo‐production. Under illumination, sulfur‐deprivation has been shown to accommodate the production of hydrogen gas by partially‐deactivating O₂ evolution activity, leading to anaerobiosis in a sealed culture. As these facets are coupled, and the system complex, mathematical approaches potentially are of significant value since they may reveal improved or even optimal schemes for maximizing hydrogen production. Here, a mechanistic model of the system is constructed from consideration of the essential pathways and processes. The role of sulfur in photosynthesis (via PSII) and the storage and catabolism of endogenous substrate, and thus growth and decay of culture density, are explicitly modeled in order to describe and explore the complex interactions that lead to H₂ production during sulfur‐deprivation. As far as possible, functional forms and parameter values are determined or estimated from experimental data. The model is compared with published experimental studies and, encouragingly, qualitative agreement for trends in hydrogen yield and initiation time are found. It is then employed to probe optimal external sulfur and illumination conditions for hydrogen production, which are found to differ depending on whether a maximum yield of gas or initial production rate is required. The model constitutes a powerful theoretical tool for investigating novel sulfur cycling regimes that may ultimately be used to improve the commercial viability of hydrogen gas production from microorganisms. Biotechnol. Bioeng. 2014;111: 320–335. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Studies on the respiration rate of free and immobilized cells of Cephalosporium acremonium in cephalosporin C production

Bioprocesses using filamentous fungi immobilized in inert supports present many advantages when compared to conventional free cell processes. However, assessment of the real advantages of the unconventional process demands a rigorous study of the limitations to diffusional mass transfer of the reagents, especially concerning oxygen. In this work, a comparative study was carried out on the cephalosporin C production process in defined medium containing glucose and sucrose as main carbon and energy sources, by free and immobilized cells of Cephalosporium acremonium ATCC 48272 in calcium alginate gel beads containing alumina. The effective diffusivity of oxygen through the gel beads and the effectiveness factors related to the respiration rate of the microorganism were determined experimentally. By applying Monod kinetics, the respiration kinetics parameters were experimentally determined in independent experiments in a complete production medium. The effectiveness factor experimental values presented good agreement with the theoretical values of the approximated zero‐order effectiveness factor, considering the dead core model. Furthermore, experimental results obtained with immobilized cells in a 1.7‐L tower bioreactor were compared with those obtained in 5‐L conventional fermentor with free cells. It could be concluded that it is possible to attain rather high production rates working with relatively large diameter gel beads (ca. 2.5 mm) and sucrose consumption‐based productivity was remarkably higher with immobilized cells, i.e., 0.33 gCPC/kg sucrose/h against 0.24 gCPC/kg sucrose/h in the aerated stirred tank bioreactor process. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 593–600, 1999.