R.G.J.M. van der Lans

Physiological responses to mixing in large scale bioreactors

Escherichia coli fed-batch cultivations at 22 m3 scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor. Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones. It is suggested that the reduced biomass yield at large scale partly is due to repeated production/re-assimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation. The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes. The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor. Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone. It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor. Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor.

Technologies for butanol recovery integrated with fermentations

Abstract In-situ product recovery can improve the performance of a butanol fermentation. Five technologies for in-situ product recovery have been compared on the basis of design parameters and energy efficiency. These five technologies are: stripping, adsorption, liquid—liquid extraction, pervaporation and membrane solvent extraction. From these, pervaporation and liquid—liquid extraction are considered to have the greatest potentials.

A simple hydrodynamic model for the liquid circulation velocity in a full-scale two- and three-phase internal airlift reactor operating in the gas recirculation regime

For design purposes a simple model to predict the hydrodynamic behaviour of a three-phase internal airlift reactor is developed. The model predicts liquid circulation, gas hold-up and minimum gas supply rates for solids suspension. The reactor type considered has an internal riser and is not equipped with a gas disengagement area, resulting in a high downcomer gas flow rate. Its purpose is waste water treatment using biofilm particles. First it is recognized that several flow regimes may be distinguished, necessitating different models. Modelling for the gas recirculation regime is straightforward based on the momentum balance combined with a simple assumption for the gas hold-up that is possible for these type of airlift reactors with unrestricted gas carryover into the downcomer. The predictive model is compared successfully with a pilot-scale reactor (400 l) and a full-scale reactor (284 m3, both with a draught tube height of about 12 m) containing up to 250 g/l solid particles with superficial gas supply rates up to 0.1 m/s.

Effects of dissolved oxygen tension and mechanical forces on fungal morphology in submerged fermentation

The effects of dissolved oxygen tension and mechanical forces on fungal morphology were both studied in the submerged fermentation of Aspergillus awamori. Pellet size, the hairy length of pellets, and the free filamentous mycelial fraction in the total biomass were found to be a function of the mechanical force intensity and to be independent of the dissolved oxygen tension provided that the dissolved oxygen tension was neither too low (5%) nor too high (330%). When the dissolved oxygen concentration was close to the saturation concentration corresponding to pure oxygen gas, A. awamori formed denser pellets and the free filamentous mycelial fraction was almost zero for a power input of about 1 W/kg. In the case of very low dissolved oxygen tension, the pellets were rather weak and fluffy so that they showed a very different appearance. The amount of biomass per pellet surface area appeared to be affected only by the dissolved oxygen tension and was proportional to the average dissolved oxygen tension to the power of 0.33. From this it was concluded that molecular diffusion was the dominant mechanism for oxygen transfer in the pellets and that convection and turbulent flow in the pellets were negligible in submerged fermentations. The biomass per wet pellet volume increased with the dissolved oxygen tension and decreased with the size of the pellets. This means that the smaller pellets formed under a higher dissolved oxygen tension had a higher intrinsic strength. Correspondingly, the porosity of the pellets was a function of the dissolved oxygen tension and the size of pellets. Within the studied range, the void fraction in the pellets was high and always much more than 50%.

Effect of Agitation Intensities on Fungal Morphology of Submerged Fermentation

Both parallel fermentations with Aspergillus awamori (CBS 115.52) and a literature study on several fungi have been carried out to determine a relation between fungal morphology and agitation intensity. The studied parameters include hyphal length, pellet size, surface structure or so-called hairy length of pellets, and dry mass per-wet-pellet volume at different specific energy dissipation rates. The literature data from different strains, different fermenters, and different cultivation conditions can be summarized to say that the main mean hyphal length is proportional to the specific energy dissipation rate according to a power function with an exponent of -0.25 +/- 0.08. Fermentations with identical inocula showed that pellet size was also a function of the specific energy dissipation rate and proportional to the specific energy dissipation rate to an exponent of -0.16 +/- 0.03. Based on the experimental observations, we propose the following mechanism of pellet damage during submerged cultivation in stirred fermenters. Interaction between mechanical forces and pellets results in the hyphal chip-off from the pellet outer zone instead of the breakup of pellets. By this mechanism, the extension of the hyphae or hair from pellets is restricted so that the size of pellets is related to the specific energy dissipation rate. Hyphae chipped off from pellets contribute free filamentous mycelia and reseed their growth. So the fraction of filamentous mycelial mass in the total biomass is related to the specific energy dissipation rate as well.To describe the surface morphology of pellets, the hyphal length in the outer zone of pellets or the so-called hairy length was measured in this study. A theoretical relation of the hairy length with the specific energy dissipation rate was derived. This relation matched the measured data well. It was found that the porosity of pellets showed an inverse relationship with the specific energy dissipation rate and that the dry biomass per-wet-pellet volume increased with the specific energy dissipation rates. This means that the tensile strength of pellets increased with the increase of specific energy dissipation rate. The assumption of a constant tensile strength, which is often used in literature, is then not valid for the derivation of the relation between pellet size and specific energy dissipation rate. The fraction of free filamentous mycelia in the total biomass appeared to be a function of the specific energy dissipation in stirred bioreactors. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 715-726, 1997.

Butanol recovery from fermentations by liquid-liquid extraction and membrane solvent extraction

Extraction can successfully be used for in-situ alcohol recovery in butanol fermentations to increase the substrate conversion. An advantage of extraction over other recovery methods may be the high capacity of the solvent and the high selectivity of the alcohol/water separation. Extraction, however, is a comprehensive operation, and the design of an extraction apparatus can be complex. The aim of this study is to assess the practical applicability of liquid-liquid extraction and membrane solvent extraction in butanol fermentations. In this view various aspects of extraction processes were investigated.Thirty-six chemicals were tested for the distribution coefficient for butanol, the selectivity of alcohol/water separation and the toxicity towards Clostridia. Convenient extractants were found in the group of esters with high molar mass.Liquid-liquid extraction was carried out in a stirred fermenter and a spray column. The formation of emulsions and the fouling of the solvent in a fermentation broth causes problems with the operation of this type of equipment. With membrane solvent extraction, in which the solvent is separated from the broth by a membrane, a dispersion-free extraction is possible, leading to an easy operation of the equipment. In this case the mass transfer in the membrane becomes important.With membrane solvent extraction the development of a process is emphasized in which the extraction characteristics of the solvent are combined with the property of silicone rubber membranes to separate butanol from water. In the case of apolar solvents with a high molar mass, the characteristics of the membrane process are determined completely by the solvent. In the case of polar solvents (e.g. ethylene glycol), the permselectivity of the membrane can profitably be used. This concept leads to a novel type of extraction process in which alcohol is extracted with a water-soluble solvent via a hydrophobic semipermeable membrane. This extraction process has been investigated for the recovery of butanol and ethanol from water. A major drawback in all processes with membrane solvent extraction was the permeation of part of the solvent to the aqueous phase.The extraction processes were coupled to batch, fed batch and continuous butanol fermentations to affirm the applicability of the recovery techniques in the actual process. In the batch and fed batch fermentations a three-fold increase in the substrate consumption could be achieved, in the continuous fermentation about 30% increase.

Modeling and measurements of fungal growth and morphology in submerged fermentations

Generalizing results from fungal fermentations is difficult due to their high sensitivity toward slight variation in starting conditions, poor reproducibility, and difference in strains. In this study a mathematical model is presented in which oxygen transfer, agitation intensity, dissolved oxygen tension, pellet size, formation of mycelia, the fraction of mycelia in the total biomass, carbohydrate source consumption, and biomass growth are taken into account. Two parameters were estimated from simulation, whereas all others are based on measurements or were taken from literature. Experimental data are obtained from the fermentations in both 2 L and 100 L fermentors at various conditions. Comparison of the simulation with experiments shows that the model can fairly well describe the time course of fungal growth (such as biomass and carbohydrate source concentrations) and fungal morphology (such as pellet size and the fraction of pellets in the total biomass). The model predicts that a stronger agitation intensity leads to a smaller pellet size and a lower fraction of pellets in the total biomass. At the same agitation intensity, pellet size is hardly affected by the dissolved oxygen tension, whereas the fraction of mycelia decreases slightly with an increase of the dissolved oxygen tension in the bulk. All of these are in line with observations at the corresponding conditions.

Effects of the aeration rate on the fermentation of glucose and xylose by Pichia stipitis CBS 5773

Abstract The effects of medium composition and aeration on the fermentation of xylose and glucose by Pichia stipitis CBS 5773 were investigated. The addition of yeast extract (5 kg m 3 ) has a positive effect on the growth of P. stipitis . There is a linear relation between the oxygen uptake and the biomass production. The bruto yield of biomass on oxygen ( Y ″ ox ) is 39.5 g mol −1 for growth on xylose and is dependent on the oxygen supply rate for growth on glucose. Under restricted aeration the process is determined by the oxygen supply. A negative correlation has been found between the yield of ethanol on substrate and the oxygen uptake. The biomass yield increases with increasing oxygen uptake. A low aeration rate is necessary for an optimal conversion of glucose and xylose to ethanol. The maximum specific ethanol production rates on glucose and xylose were respectively 0.35 and 0.13 g g −1 h −1 . This maximum was reached at oxygen uptake rates below 0.005 mol l −1 h −1 . Due to a limited substrate uptake rate, a high cell concentration will be needed to obtain high volumetric productivities.

Bubble recirculation regimes in an internal-loop airlift reactor

Abstract Bubble recirculation regimes have been studied in an internal-loop airlift reactor with two phases (water and air) and three phases (water, air, and polystyrene particles). The airlift was operated both in batch mode and with throughflow. In batch mode with increasing superficial gas velocity three regimes may be defined, depending on the state of gas phase in the downcomer: (1) no air bubbles, (2) bubbles remain stationary and (3) bubbles flow downwards and into the riser. In this paper the transition of regime 2 to regime 3 is studied. This transition is reflected by the increase of (i) the difference between gas holdups in the riser and downcomer and (ii) the liquid velocity in the downcomer. In regime 2 these two parameters are constant, while in regime 3 they increase with increasing superficial gas velocity. The ratio between the gas holdups in downcomer and riser is usually reported to be constant. However, in this work it is shown that the ratio is not constant and depends on the regime with parameters based on the regime transition. In regime 2 the gas holdups in riser and downcomer increase at the same rate, and in regime 3 the gas holdup in the riser increases faster than in the downcomer. In throughflow mode the regimes were less discernible. The presence of solids resulted in a decrease of the gas holdups, and lower values of the ratio between gas holdups in downcomer and riser.

Batch and continuous butanol fermentations with free cells: integration with product recovery by gas-stripping

SummaryIn a butanol batch fermentation the substrate consumption was increased threefold using in-situ product recovery by gas-stripping, in comparison with a control fermentation without product recovery. In a continuous fermentation in-situ recovery led to an increase in the biomass concentration, resulting in a threefold increase in productivity. The substrate consumption was increased by 10%. An external stripper was used as apparatus for the butanol recovery.