Dispersions in hydrocarbon fermentation. A retrospective study

A nondimensionalized plot, obtained by normalizing the drop-size distribution in the hydrocarbon phase using the Sauter mean diameter, shows a tendency towards self-preservation of the distribution. Changes of distribution in time during the course of fermentation, initial dispersed phase fraction, speed of rotation, and reactor size were taken into account. Using this self-preserving property, an empirical (single parameter) equation has been proposed for drop-size distribution. Data, available from the literature, are presented for non-biological and biological systems (gas-oil, n-hexadecane, and n-hexadecane dissolved in dewaxed gas oil as dispersed phases). The parameter, Sauter mean diameter, has been correlated with the operating conditions, and a critical review presented. Cell density was found to have significant effect on Sauter mean diameter. This effect has also been empirically explained. The possibilities of using generalized distribution in predicting the performance of fermenters is outlined.     

Study of drop and bubble sizes in a simulated mycelial fermentation broth of up to four phases

The mean sizes and size distributions of air bubbles and viscous castor oil drops were studied in a salt-rich aqueous solution (medium), first separately, and then simultaneously as a three-phase system. The dispersion was created in a 150-mm-diameter stirred tank equipped with a Rushton turbine, and the sizes were measured using an advanced video technique. Trichoderma harzianum biomass was added in some experiments to study the effect of a solid phase under unaerated and aerated conditions to give either three-or four-phase systems. In all cases, the different dispersed phases could be clearly seen. Such photoimages have never been obtained previously. For the three phases, air-oil-medium, aeration caused a drastic increase in Sauter mean drop diameter, which was greater than could be accounted for by the reduction in energy dissipation on aeration. Also, as in the unaerated case, larger drops were observed as the oil content increased. On the other hand, mean bubble sizes were significantly reduced with increasing oil phase up to 15% with bubbles inside many of the viscous drops. With the introduction of fungal biomass of increasing concentration (0.5 to 5 g L(-1)) under unaerated conditions, the Sauter mean drop diameter decreased. Finally, in the four-phase system (oil [10%]-medium-air-biomass) as found in many fermentations, all the phases (plus bubbles in drops) could clearly be seen and, as the biomass increased, a decrease in both the bubble and the drop mean diameters was found. The reduction in size of bubbles (and therefore increase in interfacial area) as the oil and bio- mass concentration increased provides a possible explanation as to why the addition of an oil phase has been reported to enhance oxygen transfer during many fermentations.     

Oil drop size and gas hold-up in an oil–water airlift system with motionless mixers

The degree of emulsification, measured as surface area of oil generated, was studied. The effect of interfacial tension, volume fraction of oil, and power per unit volume on the Sauter mean diameter of the oil drops was determined in an airlift system with motionless mixers. A mathematical expression to predict the Sauter mean diameter was developed using regression techniques. From this equation another equation, which will predict the surface area of oil in terms of the same variables, was derived. The effects of water air surface tension and power per unit volume on the gas hold-up were obtained using similar techniques. The results show that the interfacial tension and the surface tension are important variables when hydrocarbon fermentations are carried out in airlift systems.     

Growth models of cultures with two liquid phases. IV. Cell adsorption, drop size distribution, and batch growth

In this work, a mathematical model which can be used to describe butch growth in fermentations with two liquid phases present is developed for systems in which the growth limiting substrate is dissolved in the dispersed phase. The model takes into account the drop size distribution, the rate of adsorption of cells on the drop surface, the rate of desorption of cells from the drop surface, substrate transport between phases, phase equilibrium, and growth kinetics. The model also considers the effect, of coalescence and redispersion of oil drops in the system. It is assumed that the composition of the dispersed phase is such that substrate utilization from it causes little or no change in the interfacial area. A discrete uniform distribution and a discrete normal distribution which is obtained from an experimental distribution curve are used as drop size distributions. Simulation results are obtained for a wide range of parameter values using the IBM S/360 Continuous System Modeling Program.     

Oil/water and pre-emulsified oil/water (PIT) dispersions in a stirred vessel: Implications for fermentations

This study examines dispersions of rapeseed oil (RSO) in water by mechanical agitation under conditions mimicking those found in certain antibiotic fermentations; for example, in the presence of air, antifoam, and finely divided CaCO(3) particles. A problem with residual oil has been reported for such fermentations, and it has been suggested that the use of pre-emulsified oil can reduce this problem. Hence, the dispersion of a pre-emulsified oil produced by the "phase inversion temperature (PIT) method" has been evaluated. In both cases, the volume fraction of oil was 2%. For the RSO systems, a relatively high agitation speed was required to disperse the oil, especially in the presence of the particles and, when the agitation was stopped, separation occurred rapidly. The Sauter mean drop diameters depended on the system, being at an average energy dissipation rate of approximately 0.9 W kg(-1), 180 microm for RSO/water, 130 microm for RSO/water(antifoam)/air, 580 microm for RSO/water/CaCO(3), and 850 microm for RSO/water(antifoam)/air/CaCO(3). For the same four systems, the PIT emulsion, once dispersed, was very stable and the drop size was essentially independent of the operating conditions, with a Sauter mean diameter of approximately 0.3 microm. The implications of these findings for fermentations in which oil is used as a carbon source are assessed.     

Growth models of cultures with two liquid phases. III. Continuous cultures

Some mathematical models, which have been used to describe batch growth in fermentations with two liquid phases present, are used to predict the behavior of continuous fermentations in a chemostat. Two types of dispersed systems are considered in this investigation. In the first, type, it is assumed that the composition of the dispersed phase is such that, increased substrate utilization results in a decreased substrate concentration with no change in the interfacial area. In the second type of system, the dispersed phase is assumed to be pure substrate; therefore, the substrate concentration in the dispersed phase remains constant but the interfacial area is affected by changes in dilution rate. Three special cases are examined for each type of system in order to examine the effect of the interfacial area, the phase equilibrium constant, and the mass transfer coefficient on system performance. Comparison of two of the models with available experimental data shows fair agreement, between model and data.     

Characteristics of hydrocarbon uptake in cultures with two liquid phases

In hydrocarbon fermentation, the efficiency of hydrocarbon uptake by cells ins one of the keys to the economical production of single-cell protein. This work is concerned with characterization of cultures with two liquid phases for understanding the hydrocarbon uptake process by cells. Batch cultivation of Candida lipolytica was carried out in shaking flasks and in a tower fermentor with motionless mixers. Micorscopic observation and cell and hydrocarbon concentration distribution in batch cultivation showed that some cells are attached to the large oil drops ad others are free from them. Interfacial tension between oil and water and Sauter mean drop size decreased as cultivation proceeded. On the basis of the experimental results, the process of hydrocarbon uptake by cells is discussed.     

Growth models of cultures with two liquid phases. VII. Substrate dissolved in dispersed phase; effect of dispersed phase volume and temperature

The effects of dispersed phase volume and temperature on the batch growth of Candida lipolytica on gas oil are investigated. Growth parameters are presented for two sets of experiments. The shape of growth curves was basically similar to the system composed of n-hexadecane dissolved in dewaxed gas oil, in spite of the complex nature of the substance. All of the batch growth curves exhibited a linear growth region. The rate of linear growth and its length varied with change in dispersed phase volume. The effect of temperature on growth rate was investigated for temperatures ranging from 23°C to 34°C. The results show a smaller activation energy during linear growth than during the early stages of batch growth. These results are analyzed from the viewpoint of growth models presented previously. The results indicate that growth at drop surfaces is important and that segregation effects may be important.     

Batch kinetics and oxygen consumption ofCandida lipolytica 4-1 onn-alkanes

Detailed batch kinetics ofCandida lipolytica 4-1 onn-hexadecane for varying dispersed phase volume from 0.5 to 5% v/v is presented. All batch growth curves exhibited a linear growth region, indicating a substrate uptake limit. System productivities derived from the linear region were correlated to the dispersed phase volume. The correlation coefficient was identical with that obtained on gas oil. This implies that a correlation coefficient of interfacial area to the dispersed phase volume is identical for both systems. Dissolved oxygen profile and uptake of oxygen from gas phase were also measured. The oxygen uptake rate, volumetric oxygen transfer rate and oxygen demand (requirement) were calculated by means of the balance method. Under limiting dissolved oxygen concentration the maximal oxygen transfer of the fermenter was assessed.     

Oil and air dispersion in a simulated fermentation broth as a function of mycelial morphology

The culture conditions of a multiphase fermentation involving morphologically complex mycelia were simulated in order to investigate the influence of mycelial morphology (Trichoderma harzianum) on castor oil and air dispersion. Measurements of oil drops and air bubbles were obtained using an image analysis system coupled to a mixing tank. Complex interactions of the phases involved could be clearly observed. The Sauter diameter and the size distributions of drops and bubbles were affected by the morphological type of biomass (pellets or dispersed mycelia) added to the system. Larger oil drop sizes were obtained with dispersed mycelia than with pellets, as a result of the high apparent viscosity of the broth, which caused a drop in the power drawn, reducing oil drop break-up. Unexpectedly, bubble sizes observed with dispersed mycelia were smaller than with pellets, a phenomenon which can be explained by the segregation occurring at high biomass concentrations with the dispersed mycelia. Very complex oil drops were produced, containing air bubbles and a high number of structures likely consisting of small water droplets. Bubble location was influenced by biomass morphology. The percentage (in volume) of oil-trapped bubbles increased (from 32 to 80%) as dispersed mycelia concentration increased. A practically constant (32%) percentage of oil-trapped bubbles was observed with pelleted morphology at all biomass concentrations. The results evidenced the high complexity of phases interactions and the importance of mycelial morphology in such processes.     

Continuous culture of Candida lipolytica on n-hexadecane

Candida lipolytica was grown continuously on n-hexadecane as the main source of carbon. A transient continuous-culture experiment was also conducted to investigate hydrocarbon-limited growth; the hydrocarbon feed flow rate was stopped for several hours and then resumed at a reduced steady-state flow rate. Interfacial tension, Sauter mean diameter, pseudosolubility, fraction of cells in the aqueous phase, oil-phase volume fraction, and cell concentration were measured to characterize the system. The microorganisms appear to utilize both the submicron drops and the microscopic drops. The effects of interfacial tension, pseudosolubility, and unoccupied interfacial area on the kinetics of hydrocarbon fermentation are discussed here. A conceptual model for hydrocarbon uptake is presented and discussed.     

Growth models of cultures with two liquid phases. II. Pure substrate in dispersed phase

Mathematical models which can be used to describe batch frowth in fermentations with two liquid phases are developed for systems in which the growth limiting substrate is the dispersed liquid phase. Three special cases are considered assuming pure substrate in the dispersed phase and a decreasing interfacial area due to substrate consumption. In the first, it is assumed that all growth occurs at the surface of the dispersed phase. In the second and third growth occurs at the interface and in the continuous phase. The second case assumes substrate equilibrium between the two phases while the third assumes substrate consumption in the continuous phase is limited by rate of substrate transport to that phase. Since the amount of growth at the interface and substrate transport to the continuous phase depend on the interfacial area, two limiting cases for the decrease of interfacial area with substrate consumption are also considered in this investigation. The first and third models are compared with available experimental data.     

Growth models of cultures with two liquid phases. V. Substrate dissolved in dispersed phase—experimental observations

The effects of inoculum size, dispersed phase volume and substrate concentration on the batch growth of Candida lipolytica are investigated in a model system composed of n-hexadecane dissolved in dewaxed gas oil. Tabular values and parameters are presented for 16 different experiments. All of the batch growth curves exhibited a linear growth region with the length of the region ranging from 1.5 to 9.5 hours. The rate of linear growth varied both with change in dispersed phase volume and initial dispersed phase substrate concentration. A qualitative analysis of the results is presented and possible explanations for the observed linear growth rates are discussed.     

Influence of Yeast Flocculation on the Rate of Jerusalem Artichoke Extract Fermentation

Variations in residual sugar composition have been observed during Jerusalem artichoke extract fermentations by using Saccharomyces diastaticus NCYC 625, a flocculating yeast strain. In batch cultures, these differences were due to the inulin polymer size distribution of the extracts: measurements of enzymatic activities on different polymerized substrates have shown that the hydrolysis and fermentation yield decreased when the fructose/glucose ratio of the extract increased. Inulin hydrolysis appeared to be the limiting factor of the fermentation rate. A comparison of continuous and batch cultures with the same extract showed that fermentability differences were related to the structure and size of the yeast flocs. This led to an hydrolysis selectivity of the inulin polymers according to their size: the chemostat culture in which the floc average size was larger gave longer chained residual sugars. Received: 8 November 1999 / Accepted: 24 February 2000     

Characterization of a pilot plant airlift tower loop reactor: III. Evaluation of local properties of the dispersed gas phase during yeast cultivation and in model media

The local properties of the dispersed gas phase (gasholdup, bubble diamater, and bubble velocity) were measured and evaluated at different positions in the riser and downcomer of a pilot plant reactor and, for comparison, in a laboratory reactor. These were described in Parts I and II of this series of articles during yeast cultivation and with model media. In the riser of the pilot plant reactor, the local gas holdup and bubble velocities varied only slightly in axial direction. The gas holdup increased considerably, while the bubble velocity increased only slightly with aeration rate. The bubble size diminished with increasing distance from the aerator in the riser, since the primary bubble size was larger than the equilibrium bubble size. In the downcomer, the mean bubble size was smaller than in the riser. The mean bubble size varied only slightly, the bubble velocity was accelerated, and the gas holdup decreased from top to bottom in the downcomer. In pilot plant at constant aeration rate, the properties of the dispersed phase were nearly constant during the batch cultivation, i.e., they depended only slightly on the cell concentration. In the laboratory reactor, the mean bubble sizes were much larger than in the pilot plant reactor. In the laboratory reactor, the bubble velocities in the riser and downcomer increased, and the mean gas holdup and bubble diameter in the downcomer remained constant as the aeration rate was increased.     

Membrane formation by interfacial cross-linking of chitosan for microencapsulation of Lactococcus lactis

Lactic acid bacteria were microencapsulated within cross-linked chitosan membranes formed by emulsification/interfacial polymerization. The technique was modified and optimized to provide biocompatible conditions during encapsulation involving the use of mineral oils as the continuous phase and chitosan as the membrane material. Chitosan cross-linked with hexamethylene diisocyanate or glutaraldehyde resulted in strong membranes, with a narrow size distribution about a mean diameter of 150 mum. Cell viability and activity was demonstrated by the acidification of milk. Loss of acidification activity during microencapsulation was recovered in subsequent fermentations to levels similar to that of free cell fermentations. (c) 1993 John Wiley & Sons, Inc.     

The effect of agitation intensity on alkali-catalyzed methanolysis of sunflower oil

The sunflower oil methanolysis was studied in a stirred reactor at different agitation speeds. The measurements of drop size, drop size distribution and the conversion degree demonstrate the effects of the agitation speed in both non-reaction (methanol/sunflower oil) and reaction (methanol/KOH/sunflower oil) systems. Drop size distributions were found to become narrower and shift to smaller sizes with increasing agitation speed as well as with the progress of the methanolysis reaction at a constant agitation speed. During the methanolysis reaction, the Sauter-mean drop diameter stays constant in the initial slow reaction region, rapidly decreases during the fast reaction period and finally reaches the equilibrium level. Due to the fact that the interfacial area increases, one can conclude that the rate of reaction occurring at the interface will also be enhanced progressively. The "autocatalytic" behavior of the methanolysis reaction is explained by this "self-enhancement" of the interfacial area, due to intensive drop breakage process.     

Integration of Gas Enhanced Oil Recovery in Multiphase Fermentations for the Microbial Production of Fuels and Chemicals

In multiphase fermentations where the product forms a second liquid phase or where solvents are added for product extraction, turbulent conditions disperse the oil phase as droplets. Surface‐active components (SACs) present in the fermentation broth can stabilize the product droplets thus forming an emulsion. Breaking this emulsion increases process complexity and consequently the production cost. In previous works, it has been proposed to promote demulsification of oil/supernatant emulsions in an off‐line batch bubble column operating at low gas flow rate. The aim of this study is to test the performance of this recovery method integrated to a fermentation, allowing for continuous removal of the oil phase. A 500 mL bubble column is successfully integrated with a 2 L reactor during 24 h without affecting cell growth or cell viability. However, higher levels of surfactants and emulsion stability are measured in the integrated system compared to a base case, reducing its capacity for oil recovery. This is related to release of SACs due to cellular stress when circulating through the recovery column. Therefore, it is concluded that the gas bubble‐induced oil recovery method allows for oil separation and cell recycling without compromising fermentation performance; however, tuning of the column parameters considering increased levels of SACs due to cellular stress is required for improving oil recovery.     

Growth of yeast on n-alkanes. I. Stochastic model

In a system where yeast cells grow on n-alkanes dissolved in oil drops suspended in water, the dispersed oil phase will, in most cases, be fully segregated. This means that each drop has its own history that depends on its degree of saturation with yeast cells. This degree of saturation with yeast cells is determined by a stochastic process depending on adsorption, desorption, and cell production. Although many authors mention segregation as a phenomenon likely to occur, so far this segregation has hardly been taken into account. In this paper the interaction of the population of completely segregated oil drops with the population of yeast cells, which results in growth, is described. The consequences of the model are elucidated by the discussion of some extreme cases. The batch fermentation of hydrocarbons by yeast cell is simulated by means of a Monte Carlo procedure. Adsorption, desorption, and production of yeast cells are considered as chance processes. The history of all individual drops is recorder. The influence of the chance of desorption appears to be much larger than that of the chance of adsorption (at the investigated range). Also the size of the inoculum at the start of the process appears to have a strong influence on the course of fermentation.     

Cell Surface Measurements in Hydrocarbon and Carbohydrate Fermentations

Acinetobacter calcoaceticus was grown in 11-liter batch fermentations with hexadecane or sodium citrate as the sole source of carbon. Surface and interfacial tension measurements of the microbial broth indicated that surface-active compounds were being produced only during growth on the hydrocarbon substrate. Contact angle measurements of an aqueous drop on a smooth lawn of cells in a hexadecane bath indicated a highly hydrophobic surface of the cells in the initial stages of the hydrocarbon fermentation (120° contact angle). At this stage, the entire cell population was bound to the hydrocarbon-aqueous interface. The contact angle dropped rapidly to approximately 45° after 14 h into the fermentation. This coincided with a shift of the cell population to the aqueous phase. Thus, the cells demonstrated more hydrophilic characteristics in the later stages of the fermentation. Contact angles on cells grown on sodium citrate ranged from 18 to 24° throughout the fermentation. The cells appear to be highly hydrophilic during growth on a soluble substrate. From the contact angle and aqueous-hydrocarbon interfacial tension, the surface free energy of the cells was calculated along with the cell-aqueous and cell-hydrocarbon interfacial tension. The results of these measurements were useful in quantitatively evaluating the hydrophobic nature of the cell surface during growth on hydrocarbons and comparing it with the hydrophilic nature of the cell surface during growth on a soluble substrate.