Measurement of k(L)a by dynamic pressure method in pilot-plant fermentor

The dynamic pressure method (DPM) is used for measurement of k(L)a in a 1-m(3) pilot scale fermentor in coalescing (distilled water) and noncoalescing (0.3 M Na(2)SO(4) aqueous solution) batches. The method consists in recording oxygen concentration in a batch after a small pressure change (20 kPa) in the fermentor. The upward pressure change is brought about by temporary closing and subsequent throttling of outlet gas stream and the downward change by full reopening of the gas outlet. Absorption of pure oxygen yields the same k(L)a values as absorption of air. In noncoalescing batch, the downward k(L)a values are always higher than the upward values owing to spontaneous nucleation of bubbles. The experiments performed in a stirred cell confirm this behavior. Thus, only upward pressure change should be used for measurement. The correlation of k(L)a data measured in small (18-L) and large (1000-L) vessels based on power dissipated and superficial gas velocity are in a good agreement. Unlike the DPM, the classical dynamic methods yield, under the same conditions, excessively low values of k(L)a (the dynamic startup method) or fail to produce data at all (the dynamic method with interchange of air for N(2)). (c) 1994 John Wiley & Sons, Inc.     

Critical assessment of gassing-in methods for measuring k(l)a in fermentors

The reliability of dynamic measurement methods of k(l)a in fermentors using a step oxygen concentration change in the feed gas was tested. The tests were performed both for the original variant using the nitrogen right harpoon over left harpoon air exchange and the newly presented variant using the oxygen-enriched air (27 vol % O(2)) --> air exchange. The testing consisted in comparing k(l)a values determined from these methods with values determined from the steady-state Na(2)SO(3) feeding method and the dynamic pressure method, the reliability of which was proven earlier. The measurements were done in water (coalescent batch) and in 0.5M Na(2)SO(4) solution with and without the addition of 1 wt % carboxymethylcellulose (noncoalescent batches). It was found that in noncoalescent liquids the methods tested give extremely low k(l)a values (as low as 15% of the correct value). The methods are defective in principle irrespective of the gases used for exchange.     

Critical assessment of the steady-state Na2SO3 feeding method for kla measurement in fermentors

Important aspects of k(l)a measurement in agitated aerated vessels are briefly characterized from the standpoint of reliability of the measured data. It seems that most of the k(l)a data, based on a number of variants of the steady-state and dynamic methods in noncoalescent liquids, do not have a clear physical meaning, because they are affected by the differences between the actual driving force and the driving force assumed by the model used for its evaluation. A reliability test is given for the Na(2)SO(3) feeding steady-state method (FSM), by comparing the results of air and pure oxygen absorption in a noncoalescent liquid (0.5M Na(2)SO(4) solution) with the results obtained by the independent pressure step dynamic method (RDM). The RDM is one of a few variants of the dynamic method which gives correct k(l)a data unaffected by nonideal mixing of the gas phase in the reactor. It was found that the FSM yields correct k(l)a values only when pure oxygen is used for absorption. When air is absorbed, the FSM gives k(l)a values in the region of k(l)a > 0.1 s(-1) substantially (to 55%) lower than those for pure oxygen absorption.     

Identification of mass-transfer parameters and process simulation of SCP production process in airlift tower reactors with an external loop

A distributed parameter model for simulation of SCP-production processes in tower reactors with an outer loop was developed by considering substrate, cell, and CO(2) balances in the liquid phase, and O(2) and CO(3) balances in the ges phase and taking into account variations of dissolved oxygen concentration, pressure, and k(L)a along the column, as well as double substrate Monod kinetics. This model was used to describe the cultivation of Hansenula polymorpha in a tower-loop reactor (height 275 cm, diameter 15 cm). Parameter identification and process simulation were carried out by a hybrid computer. The variation of identified mass transfer parameters with fermentation time and operation mode is considered employing ethanol and glucose substrate, respectively. Relationships among k(L)a, substrate concentration, and superficial gas velocity were developed to facilitate the layout and simulation of pilot-plant reactors.     

Hydrodynamics and oxygen transfer in pneumatic bioreactor devices

Gas holdup and oxygen transfer studies in non-Newtonian suspensions of cellulose fibres conducted in two large (0.098 m(3) each) reactors are described. Both reactors-a bubble column and a similar internal loop airlift-were unusual in that they had rectangular cross-sections. In all cases gas holdups and k(L)a(L) declined with increasing solid concentration and, under identical conditions, the bubble column performed better than the airlift. The fluid systems used were carefully selected to represent mould fermentation broths.The behavior of true mass transfer coeffcient k(L) with changes in bubble size is discussed for these systems.     

Process-engineering characterization of small-scale bubble columns for microbial process development

Volumetric oxygen transfer rates and power inputs were estimated by a model of the formation of primary gas bubbles at the static sparger (sinter plate) of small-scale bubble columns and a common mass-transfer correlation for bubbles rising in a non-coalescent Newtonian electrolyte solution of low viscosity. Estimations were used to assess the dimensioning and possibilities of small-scale bubble column application with an height/diameter ratio of about 1. Estimations of volumetric oxygen transfer rates (<0.16 s^-1) and power inputs (<100 W m^-3) with a mean pore diameter of the static sparger of 13 µm were confirmed as function of the superficial air velocity (<0.6 cm s^-1) by measurements using an Escherichia coli fermentation medium. Small-scale bubble columns are thus to be classified between shaking flasks and stirred-tank reactors with respect to the oxygen transfer rate, but the maximum volumetric power input is more than one magnitude below the power input in shaking flasks, which is of the same order of magnitude as in stirred-tank reactors. A small-scale bubble columns system was developed for microbial process development, which is characterized by handling in analogy to shaking flasks, high oxygen transfer rates and simultaneous operation of up to 16 small-scale reactors with individual gas supply in an incubation chamber.     

Influence of very small bubbles on the dynamic kLA measurement in viscous gas–liquid systems

Experiments reveal a fraction of tiny bubbles (?1 mm) in viscous gas-liquid systems. It is plausible that the oxygen tension is these bubbles will be in equilibrium with that in the liquid within seconds. This means that, as regards to such oxygen concentration changes as occur on k_L A determination by the dynamic method, the “liquid-small bubble dispersion” can be considered as a homogeneous phase. This leads to major corrections and, therefore, the application of the dynamic k_L A method in viscous gas-liquid systems will be problematic.     

Promotion of oxygen transfer in three-phase fluidized-bed bioreactors by floating bubble breakers

The objective of the present study was to investigate a method to enhance the volumetric rate of oxygen transfer in three-phase fluidized-bed bioreactors. The rates of oxygen transfer from air bubbles to viscous liquid media were promoted by floating bubble breakers in three-phase fluidized beds operated in the bubble coalescing regime. The liquid-phase volumetric oxygen transfer coefficient has been recovered by fitting the axial dispersion model to the resultant data, and its dependence on the experimental variables, such as the gas and liquid flow rates, particle size, concentration of bubble breakers, and liquid viscosity, has been examined. The results indicate that the liquid-phase volumetric oxygen transfer coefficient can be enhanced up to 20-25%. The coefficient exhibits a maximum with respect to the volume ratio of the floating bubble breakers to the fluidized solid particles; it increases with increases in the gas and liquid flow rates and size of fluidized particles, while it decreases with an increase in the liquid viscosity. An expression has been developed to correlate the liquid-phase volumetric oxygen transfer coefficient with the experimental variables.     

Oxygen transfer and consumption in a thiosulfate oxidizing bioreactor with sulfur production

AIMS: To evaluate the contribution of oxygen transfer and consumption in a sulfoxidizing system to increase the elemental sulfur yield from thiosulfate oxidation. METHODS AND RESULTS: A 10 l thiosulfate oxidizing bioreactor with suspended cells operating under microaerophilic conditions and a separated aerator with a variable volume of 0.8--1.7 l were operated with a consortium containing mainly Thiobacillus sp. that oxidizes several sulfide species to elemental sulfur and sulfate. From the gas-liquid oxygen balance, the k(L)a was estimated under different operation conditions. A k(L)a of around 200 h(-1) favoured elemental sulfur production and can serve as scale-up criterion. It was further shown that more than 50% of the oxygen fed to the system was consumed in the aerator. CONCLUSIONS: The performance of the sulfoxidizing system can be improved by controlling oxygen transfer. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed method for the k(L)a determination was based on the oxygen balance, which incorporates the oxygen concentrations measured in the liquid in steady state, reducing the interference of the response time in the traditional non-steady state methods. This approach can be used to optimize reactors where microaerophilic conditions are desirable.     

Construction and performance of plug-in membrane inlet mass spectrometer for fermentor monitoring

An in situ sterilizable plug-in membrane inlet mass spectrometer for monitoring dissolved gases and volatiles in fermentors was constructed and tested. The design ensured a minimal distance to be traveled by analyte molecules from the bulk of the fermentation broth to the ionization chamber of the mass spectrometer. Apart from the specific cross talk due to overlapping mass peaks from different compounds, we found that carbon dioxide interfered unspecifically with all the mass peaks of other substances, changing them by the same factor. The interference changed slowly with time and could be positive or negative depending on the history of the mass spectrometer. Also, the general sensitivity of the instrument changed slowly with time. These effects can be neglected or corrected for empirically in short-term measurements. When the fermentor was aerated with a three-component gas mixture including carbon dioxide, a rapid change in the partial pressure of carbon dioxide in the gas mixture gave rise to a transient in the signal of a gas whose partial pressure was kept constant. This effect revealed a transient change in the composition of the gas mixture in the bubbles caused by net import or export of carbon dioxide during equilibration with the new gas mixture. An experimental method to determine the effective partial pressures of gases in the bubbles during steady-state transport of carbon dioxide was designed. The plug-in membrane inlet mass spectrometer was tried as a probe for oxygen and ethanol in an oxystatic culture of the yeast Pichia stipitis. We found that it was possible to keep a steady-state concentration of as little as 0.5 muM throughout the lifetime of the culture. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 535-542, 1997.     

An alternative explanation for cyanobacterial scum formation and persistence by oxygenic photosynthesis

The cause of persistent cyanobacteria scum formation in lakes is an unresolved subject. Scum refers to the event in which cyanobacteria are at the water surface of a lake. Factors like low turbulence levels, long day-light, high water temperatures and the buoyant capacity of cyanobacterial cells play a role in the occurrence of scums. However, they do not explain why scums are observed at periods during the day when according to theory they should have disappeared into the deeper water layers. In this study, we present an alternative explanation. The hypothesis we present here is that irreversible buoyancy of cyanobacteria colonies is created by the growth of gas bubbles on or within the mucilage of the colonies. These bubbles grow under oxygen super-saturated conditions. At low wind speed and high chlorophyll levels, the dissolved oxygen (DO) produced during photosynthesis by cyanobacteria, cannot escape sufficiently fast to the atmosphere hence a DO supersaturated condition arises in the water. At this stage, growth of oxygen bubbles may occur inside or attached to the mucilage. We present results of compression experiments to support our hypothesis. In a chamber, the pressure on lake water containing a natural cyanobacteria population is increased. At 3 × 10⁵ and 4 × 10⁵ Pa the cyanobacteria colonies were not able to float anymore and sank. This pressure is lower than the 10⁶ Pa needed to collapse all gas vacuoles inside the cyanobacteria cells (Walsby, 1994). The observed change from floating to sinking colonies due to increased water pressure suggests that gas bubbles were present inside the colonies. In lakes, these gas bubbles may lead to permanent buoyancy, i.e. a persistent scum.     

Oxygen transfer in a multiple air-lift loop reactor

The Multiple Air-lift Loop reactor (MAL) is a new type of bioreactor, in which a series of airlifts with internal loops is incorporated into one vessel. As such, the MAL is an approximation of an aerated plug-flow fermenter. Gas/liquid oxygen transfer was studied as a function of the gas flow rate in a MAL. The second MAL-compartment in the series was investigated in particular, and a Rectangular Air-lift Loop reactor (RAL) was used as a reference. Both a dynamic and a steady-state method were used for the determination of the overall volumetric oxygen-transfer coefficient. Both methods gave the same results. The oxygen transfer coefficient in the second MAL-compartment was low compared to that of conventional internal-loop reactors. Wall effects probably caused bubble coalescence and a reduction in the oxygen transfer. For the RAL it was found that oxygen transfer was comparable to that in a bubble column.     

Investigation of the behavior of dissolved gases during freezing

This paper deals with the freezing process of aqueous solutions of gases and the nucleation of gas bubbles at the moving ice—water interface. A cryomicroscope was used to investigate the conditions of nucleation and growth of bubbles after reaching a stationary concentration profile ahead of the phase boundary. The enrichment of gases due to the distribution coefficient was detected by means of a test bubble method, i.e., the increase in the radius of a small bubble being approached by the ice front. A distribution coefficient of 0.048 (at 0 °C) was found for oxygen. Nucleation occurs when stationary growth conditions in the solution are reached. The measured oversaturation is close to 20, i.e., about the inverse of the distribution coefficient. In highly saturated gas solutions, dendritic breakdown of the planar ice-water interface due to gas enrichment could be observed. At these positions also a considerable degree of constitutional supercooling was found. Bubbles were nucleated in interdendritic spaces. Nucleation and growth of gas bubbles was seen to be a periodic process under certain circumstances which can be explained by the continuous buildup and reduction of the concentration field in the remaining solution. The growth kinetics of the bubbles and their maximum size are governed by the velocity of the ice-water interface. During growth the gas bubbles are pushed and partially encapsulated, until they reach a radius in the order of magnitude of the diffusion boundary layer of the concentration profile, and become totally engulfed by the solid phase.     

Hydraulic Conductivity Recovery versus Water Pressure in Xylem of Acer saccharum

Experiments were conducted to determine the influence of stem diameter, xylem pressure potential, and temperature on the rate of recovery of hydraulic conductivity in embolized stems of Acer saccharum Marsh. Recovery of conductivity was accompanied by an increase in stem water content as water replaced air bubbles and bubbles dissolved from vessels into the surrounding water. The time required for stems to go from less than 3 to 100% hydraulic conductivity increased approximately with the square of the stem diameter and increased with decreasing xylem pressure potential. Recovery was halted when xylem pressure potential decreased below −6 kPa. Increasing xylem pressure from 13 to 150 kPa reduced the time for recovery by a factor of 4. Temperature had little influence on the rate of recovery of hydraulic conductivity. All of these results are in accord with a theory of bubble dissolution in which it is assumed that: (a) the rate of bubble dissolution is rate limited by diffusion of air from the bubbles to the outer surface of the stems, (b) the equilibrium concentration of gases in liquid in stems is determined by Henry's law at all air-water interfaces, (c) the equilibrium solubility concentration is determined only by the partial pressure of the gas in the gas phase and not directly by the liquid-phase pressure, and (d) the gas pressure of an entrapped air bubble in the lumen of a cell can never be less than atmospheric pressure at equilibrium.     

Development and evaluation of stability and ultrasound response of DSPC-DPSG-based freeze-dried microbubbles

Abstract

It is known that Phosphatidyl choline-Phosphatidyl glycerol mixtures can be used for liposome formulations, making them less leaky than liposomes with only one lipid. We hypothesized that this might also be the case for bubbles, which can be used as ultrasound (US) contrast agents. Therefore, we have compared a series of mixed distearoyl phosphatidylcholine-distearoyl phosphatidylglycerol (DSPC-DPSG) bubbles and with bubbles containing either DSPC or DSPG (and distearoyl ethanolamine-polyethyleneglycol 2000, DSPE-PEG2k). Here, we describe the development, examination of stability in vitro and attenuation of broad frequency US pulses. Novel lipid-stabilized freeze-dried formulations for US applications, using the phospholipids DSPC, DSPG, and PEGylated DSPE-PEG2k and perfluoropropane gas were developed. It was found that the bubbles could effectively be preserved by freeze-drying and then re-constituted by addition of water. Average bubble sizes were around 2?µm for all bubbles after re-constitution. Bubble stability was assessed by evaluating the decay of the US backscattering signal in vitro. Bubbles containing DSPG were more stable than bubbles with only DSPC. The composition DSPC:DSPG:DSPE-PEG2k 30:60:10 (molar ratio) was the most stable with an effective half-life of 9.12?min, compared to bubbles without DSPG, which had half-life of 2.05?min. Bubble attenuation of US depended highly on the compositions. Bubbles without DSPG had the highest attenuation indicating higher oscillation the most but were also destroyed by higher energy US. No bubbles with DSPG showed any indication of destruction but all had increased attenuations to varying degrees, DSPC:DSPG:DSPE-PEG2k 45:45:10 showed the least attenuation.     

Incorporating hydrodynamics into spatiotemporal metabolic models of bubble column gas fermentation

Gas fermentation has emerged as a technologically and economically attractive option for producing renewable fuels and chemicals from carbon monoxide (CO) rich waste streams. LanzaTech has developed a proprietary strain of the gas fermentating acetogen Clostridium autoethanogenum as a microbial platform for synthesizing ethanol, 2,3-butanediol, and other chemicals. Bubble column reactor technology is being developed for the large-scale production, motivating the investigation of multiphase reactor hydrodynamics. In this study, we combined hydrodynamics with a genome-scale reconstruction of C. autoethanogenum metabolism and multiphase convection–dispersion equations to compare the performance of bubble column reactors with and without liquid recycle. For both reactor configurations, hydrodynamics was predicted to diminish bubble column performance with respect to CO conversion, biomass production, and ethanol production when compared with bubble column models in which the gas phase was modeled as ideal plug flow plus axial dispersion. Liquid recycle was predicted to be advantageous by increasing CO conversion, biomass production, and ethanol and 2,3-butanediol production compared with the non-recycle reactor configuration. Parametric studies performed for the liquid recycle configuration with two-phase hydrodynamics showed that increased CO feed flow rates (more gas supply), smaller CO gas bubbles (more gas–liquid mass transfer), and shorter column heights (more gas per volume of liquid per time) favored ethanol production over acetate production. Our computational results demonstrate the power of combining cellular metabolic models and two-phase hydrodynamics for simulating and optimizing gas fermentation reactors.     

Bubble bed reactor: A reactor design to minimize the damage of bubble aeration on animal cells

A new bubble aeration system was designed to minimize cell killing and cellular damage due to sparging. The residence time of the bubbles in the developed bubble bed reactor was prolonged dramatically by floating them in a countercurrent produced by an impeller. The performance of the new reactor bubble aeration system, implemented in a laboratory reactor, was tested in dynamic aeration experiments with an without cells. An efficiency up to 95% in oxygen transfer could be achieved, which enables a much lower gas flow rate compared with conventional bubble aeration reactors. The low gas flow rate is important to keep cell damage by bubbles as low as possible. A laser light sheet technique used to find the optimal flow pattern in the reactor. The specific power dissipation of the impeller is a good measure to predict cell damage in a turbulent flow. Typical values for the power dissipation measured in the bubble bed reactor were in the range of 0.002 to 0.013 W/kg, which is far below the critical limit for animal cells. The growth of a hybridoma cell line was studied in cell cultivation experiments. A protein-free medium without supplements such as serum or Pluronic F68 was used to exclude any effect of cell-protecting factors, No difference in the specific growth rate and the yield of the antibodies was observed in cell grown in the bubble free surface aeration in the spinner flask. In contrast to the spinner flask, however, the bubble bed reactor design could be scaled up. (c) 1994 John Wiley & Sons, Inc.     

Oxygen mass transfer into aerated CMC solutions in a bubble column

Differing findings on the volumetric mass transfer coefficients k(L)a in CMC solutions in bubble column bioreactors have been reported in the literature. Therefore, oxygen mass transfer was studied again in CMC solutions in a 14-cm-i.d. x 270-cm-height bubble column using different spargers. The k(L)a values were determined along with the dispersion coefficients by fitting the prediction of the axial dispersed plug model with the experimental oxygen concentration profiles in the liquid phase. Surprisingly, the obtained liquid phase dispersion coefficients for CMC solution are higher than one would expect from correlations. The k(L)a data depend largely on the flow regime. In general, they are lower than those reported in the literature. The data for developing slug and established slug flow are dependent on the gas velocity and the effective viscosity of the solution and can br correlated by a simple correlation. This correlation describes k(L)a values measured on fermentation broth of Penicillium chrysogenum with striking agreement.     

Studies on automatically aerated biosynthetic processes. II. Occurrence and elimination of CO2 during penicillin biosynthesis

Oxygen uptake of Penicillium chrysogenum hyphae growing in automatically aerated deep cultures was the subject of local and periodical change. The change depended on the concentration of carbon dioxide which accumulated in the gas phase of system during the evolution of foam bubbles, and which was suddenly liberated when the foam was destroyed. The actual concentration of sunflower oil added as an antifoaming agent also influenced the oxygen uptake of culture.