Effect of fermentation broth and biosurfactants on mass transfer during liquid-liquid extraction

Mass transfer rates in liquid-liquid extraction processes can be seriously affected by the presence of surface-active contaminants. This is especially true of applications of a biotechnological origin, where the microorganism used in the process may produce the surface-active contaminants. An investigation into the effects of soluble and insoluble fermentation broth components on mass transfer using chloramphenicol extraction into octanol as the model system was conducted. Soluble components produced during fermentation were found to adsorb to the interface, where they reduced the overall mass transfer coefficient by up to 70%. After fractionation it was found that components in the weight range from 10-30 kDa had the greatest effect on mass transfer. Protein and phospholipid compounds of similar size were found to reduce the overall mass transfer coefficient to a similar extent to the broth components at concentrations around 0.001mg/l. The biomass produced during the fermentation also reduced mass transfer substantially, and it is likely that this was due to physical blockage of the interface.     

Coextraction during reactive extraction of phenylalanine using Aliquat 336: modeling extraction equilibrium

Reactive liquid-liquid extraction can be used to recover hydrophilic fermentation products that would not otherwise partition into nonpolar solvents through an ion-exchange reaction at the two-phase interface. However, the ion-exchange reagent may not be specific to the solute of interest and other compounds present may also be extracted. In this study, the effect on solute extraction of other compounds present in the extraction medium was investigated for phenylalanine extraction using Aliquat 336. The extent of extraction at equilibrium was modeled using the equilibrium constants for the reactions present in the process. The interaction of different species within a multicomponent medium was examined using the model and experimental results. It was found that the extent of extraction and coextraction is controlled by the thermodynamics of each extraction reaction and, due to the formation of a common product, the interaction between each of the reactions. The main competition to reactive extraction will come from hydrophobic anionic compounds that will be present in fermentation broth.     

Passive Asymmetric Transport through Biological Membranes

The magnitude of passive diffusional solute transfer through artificial membranes is usually considered to be independent of the direction of the concentration gradient driving force. It can be shown, however, that a composite membrane, having as one component a membrane with a chemical reaction-facilitated diffusion transport mechanism, can result in an asymmetrical flux. An asymmetric flux caused by this type of structural heterogeneity may be one mechanism contributing to the asymmetric properties of biological membranes. Similar vectorial fluxes can be generated in interfacial solute transfer through membranes if hydrodynamic boundary layers occur at the membrane interface and reversible chemical reactions with the permeant species are involved in either phase.     

Interactions between surfactants and biomass during liquid–liquid extraction

Fermentation systems can contain may surface‐active compounds that can interfere with downstream separation processes. This work examines the interactions that can occur between surfactants and biomass during solute mass transfer in a liquid–liquid extraction system. Adding the surfactants sodium dodecyl sulfate and dodecyl trimethyl ammonium bromide to the aqueous phase caused a substantial increase in the mass transfer of chloramphenicol between water and octanol. Further investigation of the interfacial region using an optical Schlieren apparatus revealed that these increases were due to interfacial turbulence that gave rise to a rapid surface renewal convective mass transfer mechanism. When microbial biomass was present with sodium dodecyl sulfate, an increase in the mass transfer rate was again found, however, to a lesser extent. In contrast, dodecyl trimethyl ammonium bromide did not promote mass transfer and it is postulated that electrical interactions between the surfactant and the cell surface prevented adsorption of either at the interface. The interaction between the antifoaming agent polypropylene glycol 2000 and extraction system components was also investigated, with both positive and negative effects being recorded under varying conditions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Effect of microorganisms on rate of liquid extraction of ethanol from fermentation broths

Liquid extraction is one means of removing metabolic products continuously during a fermentation and so reducing product inhibition. It is known that microbial organisms are attracted to liquid-liquid interfaces, and it is important for the design of extraction systems to establish if this has a detrimental effect on the rate of extraction. The extraction of ethanol from aqueous suspensions of yeast (Saccharomyces cerevisiae) using n- decanol is described in this paper. It was found that the presence of the yeast cells severely reduced the rate of ethanol extraction. The overall mass transfer coefficient was reduced from 5.0 x 10(-6) to 0.7 x 10(-6) m/s. This reduced overall mass transfer coefficient was unaffected by yeast concentration in the range 0.1-20 kg/m(3). The results are consistent with the yeast cells adsorbing to the interface in closely packed layers and preventing mass transfer by simply reducing the available interfacial area. Optical microscope observations confirmed that a yeast layer several cell diameters thick rapidly built up at the interface when a small decanol droplet was added to a yeast suspension.     

Emulsion stability and demulsification during the direct extraction of penicillin broth

In this article, we present the factors which influence the stability of the emulsion forming under the circumstances of the direct extraction of penicillin fermentation broth and the conditions of demulsification. A laboratory method has been developed for testing demulsifiers and a quantitative evaluation of the properties determining the separation of phases has been worked out as well. It has been found that an emulsion of oil in water (o/w) forms and that this is very stable; it can be broken only by the presence of demulsifier and by using a field of centrifugal force. We have discovered that the most effective demulsifiers are cation-active surfactants for 1-4 g/L broth concentration. Furthermore, it is advantageous if the demulsifier contains a nonionic surfactant and wetting agent as well. From the 19 surface-active compounds studied in the laboratory, we found that the NADAR 107 A and ARMOGARD D-5306 demulsifiers contained the best properties for the extraction of the penicillin broth.     

Measurement of interfacial areas in aerobic fermentations by ultrasonic pulse transmission

The penetration of ultrasonic waves through opaque media and the large difference in the acoustic properties between air bubbles and the fermentation broth were used to measure the energy attenuation of pulsed ultrasound by the bubbles as the waves passed through the broth. This leads to an on-line determination of the specific interfacial area provided information is available about the holdup or bubble mean diameter. This article gives the principle of the method and demonstrates how the measured interfacial area may be used in evaluating the mass transfer coefficient of a fermentation system in a bubble column.     

New reactive extraction systems for separation of bio-succinic acid

Biotechnologically produced succinic acid has the potential to displace maleic acid and its uses and to become an important feedstock for the chemical industry. In addition to optimized production strains and fermentation processes, an efficient separation of succinic acid from the aqueous fermentation broth is indispensable to compete with the current petrochemical production processes. In this context, high molecular weight amines are known to be effective extractants for organic acids. For this reason, as a first step of isolation and purification, the reactive extraction of succinic acid was studied by mixing aqueous succinic acid solutions with 448 different amine–solvent mixtures as extraction agents (mixer-settler studies). The extraction agents consist either of one amine and one solvent (208 reactive extraction systems) or two amines and two solvents (240 reactive extraction systems). Maximum extraction yields of succinic acid from an aqueous solution with 423 mM succinic acid at pH 2.0 were obtained with more than 95% yield with trihexylamine solved in 1-octanol or with dihexylamine and diisooctylamine solved in 1-octanol and 1-hexanol. Applying these optimized reactive extraction systems with Escherichia coli fermentation broth resulted in extraction yields of 78–85% due to the increased ionic strength of the fermentation supernatant and the co-extraction of other organic acids (e.g., lactic acid and acetic acid), which represent typical fermentation byproducts.     

Extraction of penicillin-G by aqueous two-phase partition

Summary Pure potassium penicillin-G solution and penicillin-G fermentation broth were extracted using the aqueous two-phase systems of poly-ethylene glycol and salts. The partition coefficients of penicillin-G were above 10 in both pure solution and broth systems. The partition coefficients of phenyl acetic acid were about 0.25. Cell mass and solid residue in the broth system were partly concentrated around the interfacial region and partly settled to the bottom when under 1000 × g centrifugal force. Accordingly, this technique is a promising alternative for the recovery of penicillins.     

Preferential orientation of an immunoglobulin in a glycolipid monolayer controlled by the disintegration kinetics of proteo-lipidic vesicles spread at an air-buffer interface

The insertion of immunoglobulin (IgG) in a glycolipid monolayer was achieved by using the ability of new proteo-glycolipid vesicles to disintegrate into a mixed IgG-glycolipid interfacial film after spreading at an air-buffer interface. The interfacial disintegration kinetics was shown to be directly dependent on the initial vesicle surface density and on the buffer ionic strength. The presence of the immunoglobulin in the glycolipid film was displayed by an increase of the lateral compressibility (Cs) during monolayer compression. Cs magnitude modifications, due to the antibody effect on the monolayer packing, decreases as the spread vesicle density increases. At interfacial saturation, the lateral compressibility profile becomes similar to that of a control monolayer without antibody. However, the careful analysis of the mixed monolayer after transfer by Langmuir-Blodgett technique (ATR-FTIR characterisation, enzyme immunoassociation) clearly demonstrated that the antibody was still present in such conditions and was not completely squeezed out from the interface as compressibility changes could have meant. At nonsaturating vesicle surface density, IgG molecules initially lying in the lipid matrix with the Y-shape plane parallel to the interface move to a standing-up position during the compression, leading to lateral compressibility modifications. For a saturating vesicle surface density, the glycolipid molecules force the IgG molecules to directly adopt a more vertical position in the interfacial film and, consequently, no lateral compressibility modification was recorded during the compression.     

Enhancement of mass transfer using colloidal liquid aphrons: measurement of mass transfer coefficients in liquid-liquid extraction

Interphase mass transfer of a sparingly soluble solute is often the rate-limiting step in multiphase biocatalytic processes. Colloidal liquid aphrons (CLA) provide very large interfacial areas, and thus could enhance mass transfer in such processes. The aim of this study was to characterize mass transfer properties of CLA dispersions during transfer of heptanoic acid from water to limonene. The interfacial area per unit volume (a), film mass transfer coefficient (K(L)), and volumetric mass transfer coefficient (K(L)a) values were determined in a stirred-tank reactor. These results were used, along with a literature correlation, to estimate the mass transfer coefficient of the surfactant-stabilized shell surrounding the CLA. The very large increase in a provided by the CLA was only partially offset by a slight increase in the mass transfer resistance of the shell. As a result, the range of K(L)a values obtained using CLA was about an order of magnitude greater than that obtained using a conventional dispersion. The concentration of the aqueous-phase surfactant used to form the CLA strongly affected the Sauter mean diameter of the CLA; however, the concentration of the nonpolar-phase surfactant had little effect. These results suggest that CLA have considerable potential for multiphase biocatalytic applications.     

Ethanol production by extractive fermentation

The ideal method to produce a terminal metabolite inhibitor of cell growth and production is to remove and recover it from the fermenting broth as it formed. Extractive fermentation is achieved in the case of ethanol production by coupling both fermentation and liquid-liquid extraction, The solvent of extraction is 1-dodecanol (or a mixture 1-dedecanol, 1-tetradecanol); study of the inhibitory effect of primary aliphatic alcohols of different chain lengths shows that no growth is observed in the presence of alcohols which have between 2 and 12 carbons. This effect is suppressed when the carbon number is 12 or higher. A new reactor has been used-1 pulsed packed column. Pulsation is performed pneumatically. Porous material used as a package adsorbs the cells. The fermentation broth is pulsed in order to (1) increase the interfacial area between the aqueous phase and the dodecanol, (2) decrease gas holdup. Alcoholic fermentation, performed at 35 degrees C on glucose syrup, permits the total utilization of glucose solution of 409 g/L with a yeast which cannot-in classical process- completely use solutions with 200 g/L of glucose. The feasibility of a new method of fermentation coupling both liquid-liquid extraction and fermentation is demonstrated. Extension of this method is possible to any microbial production inhibited by its metabolite excretion.     

The reactive extraction of phenylalanine with Aliquat 336: buffer co-extraction equilibrium and mass transfer kinetics

The occurrence of significant co-extraction of buffer anions by the ion exchanger Aliquat 336 is unavoidable when high levels of system buffering is required. The co-extraction will result in inaccurate equilibrium and mass-transfer characterization of such a system unless its occurrence is taken into account, making process design and control difficult. A study of the equilibrium of phenylalanine extraction using Aliquat 336, a system where high levels of hydroxyl co-extraction occurs, was used as a model case to develop a method of accounting for co-extraction in mass-transfer modeling. Analysis of the equilibrium between bulk-aqueous-phase chloride and phenylalanine concentrations during mass transfer in a stirred-transfer cell showed there to be linear equilibrium relationships between the two parameters for a given extraction system of the form C(Cl,t) = alpha(C(A,t) - C(A,0)) for forward extraction and C(Cl,t) = epsilon C(A,t) + C(Cl,0) for backward extraction. The constants of proportionality of these relationships, or the "co-extraction constants," alpha and epsilon, were shown to be related to the selectivity of Aliquat 336 for the phenylalanine anion by the relationships alpha = -(1/S + 1) and epsilon; = -(1/S(-1) + 1). The linear equilibrium relationships were used to develop two-film theory mass-transfer models for both forward and backward extraction that account for co-extraction. These showed much higher accuracy in modeling stirred-transfer-cell data than the equivalent models which ignored co-extraction.     

Kinetics of the conversion of glucose to gluconic acid by Pseudomonas ovalis

The concept of a “critical oxygen concentration” is conventionally considered to hold for the submerged aerobic fermentation of glucose to gluconic acid. Above the critical level the fermentation rate is supposedly independent of oxygen concentration. In this work it is shown that, at a given agitation rate, the fermentation is independent of dissolved oxygen when above the critical. However, an increase in the agitation rate results in an increase in the fermentation rate. This increase was shown to be accompanied by an increase in the gluconolactone concentration in the broth. Gluconolactone, an intermediate in the reaction pathway, is hydrolyzed nonenzymatically to gluconic acid. Evidence is presented to suggest that the increased gas-liquid interfacial area brought about by increased agitation causes an increased net rate of lactone formation. This in turn results in an increased rate of hydrolysis of the lactone to gluconic acid. A model is presented hypothesizing that negatively charged cells adsorb at the gas-liquid interface. These cells attract hydrogen ions, causing a lowering of the pH in the film around the bubbles. It is this lowered pH which is considered to bring about increased fermentation rates when the interfacial area is increased. Supporting evidence is presented.     

Dynamic simulation of the integrated bioreactor

The article reports numerical simulation for the system combining selective adsorption of fructose and an enzyme reaction to produce higher fructose syrup. A procedure for the dynamic simulation of the process in the frame of equations for mass transfer in fixed bed, is obtained by making a solute material balance. A fixed bed model incorporating axial dispersion and a linear driving force for mass transfer was successfully used to estimate optimum operating conditions (switch times and fluid flow rates)     

Rheology and Hydrodynamic Properties of Tolypocladium inflatum Fermentation Broth and Its Simulation

A physico-chemical, two phase simulated pseudoplastic fermentation (SPF) broth was investigated in which Solka Floc cellulose fibre was used to simulate the filamentous biomass, and a mixture of 0.1% (w/v) carboxymethyl cellulose (CMC) and 0.15 M aqueous sodium chloride was used to simulate the liquid fraction of the fermentation broth. An investigation of the rheological behaviour and hydrodynamic properties of the SPF broth was carried out, and compared to both a fungal Tolypocladium inflatum fermentation broth and a CMC solution in a 50 L stirred tank bioreactor equipped with conventional Rushton turbines. The experimental data confirmed the ability of the two phase SPF broth to mimic both the T. inflatum broth bulk rheology as well as the mixing and mass transfer behaviour. In contrast, using a homogeneous CMC solution with a similar bulk rheology to simulate the fermentation resulted in a significant underestimation of the mass transfer and mixing times. The presence of the solid phase and its microstructure in the SPF broth appear to play a significant role in gas holdup and bubble size, thus leading to the different behaviours. The SPF broth seems to be a more accurate simulation fluid that can be used to predict the bioreactor mixing and mass transfer performance in filamentous fermentations, in comparison with CMC solutions used in some previous studies.     

Acetone–butanol–ethanol competitive sorption simulation from single,binary, and ternary systems in a fixed‐bed of KA‐I resin

Separation of butanol based on sorption methodology from acetone–butanol–ethanol (ABE) fermentation broth has advantages in terms of biocompatibility and stability, as well as economy, and therefore gains much attention. In this work a chromatographic column model based on the solid film linear driving force approach and the competitive Langmuir isotherm equations was used to predict the competitive sorption behaviors of ABE single, binary, and ternary mixture. It was observed that the outlet concentration of weaker retained components exceeded the inlet concentration, which is an evidence of competitive adsorption. Butanol, the strongest retained component, could replace ethanol almost completely and also most of acetone. In the end of this work, the proposed model was validated by comparison of the experimental and predicted ABE ternary breakthrough curves using the real ABE fermentation broth as a feed solution. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:124–134, 2015     

Determination of 5alpha-androst-16-en-3alpha-ol in truffle fermentation broth by solid-phase extraction coupled with gas chromatography-flame ionization detector/electron impact mass spectrometry

A novel method using solid-phase extraction coupled with gas chromatography and flame ionization detector (FID)/electron impact mass spectrometry (EIMS) was developed for the determination of 5alpha-androst-16-en-3alpha-ol (androstenol), a steroidal compound belonging to the group of musk odorous 16-androstenes, in truffle fermentation broth. Comparison studies between FID and EIMS indicated two detectors gave similar quantitative results. The highest androstenol concentration of 123.5 ng/mL was detected in Tuber indicum fermentation broth, while no androstenol was found in Tuber aestivum fermentation broth. For the first time, this work confirmed the existence of androstenol in the truffle fermentation broth, which suggested truffle fermentation is a promising alternative for androstenol production on a large scale.     

The effects of non-Newtonian fermentation broth viscosity and small bubble segregation on oxygen mass transfer in gas-lift bioreactors: a critical review

This review paper deals with the effects of non-Newtonian fermentation broth viscosities on gas–liquid mixing and oxygen mass transfer characteristics to provide knowledge for the design and development of gas-lift bioreactors, which can operate satisfactorily with high viscosity fermentation broths. The effect of small bubble segregation is also examined.     

An on-line sampling system for fermentation monitoring using membrane inlet mass spectrometry (MIMS): application to phenoxyacetic acid monitoring in penicillin fermentation

A sampling system for on-line monitoring of organic compounds of low volatility in complex fermentation media uses membrane inlet mass spectrometry (MIMS). A Syringe pump draws a continuous flow of microfiltered broth from the reactor and circulates it after acidification through a membrane inlet, in which a membrane is the only interface between the sample and the high vacuum of a mass spectrometer. All operations run automatically, i.e., sampling, acidification measurement, and calibration. The on-stream acidification enables MIMS monitoring of carboxylic acids, as they must be undissociated in order to pass the hydrophobic membrane. The performance of the monitoring system was tested by measurements of standard solutions of phenoxyacetic acid (POAA, the sie chain precursor of penicillin-V) as well as on POAA during 200 h penicillin-V fermentation. During the entire fermentation POAA was monitored n low millimolar concentrations with high accuracy and fast response to step changes in POAA concentration. Tandem mass spectrometry (MS/MS) allowed direct identification of peaks in the mass spectrum of the broth that were not accounted for by POAA. These peaks were identified as SO(2) and SCO. (c) 1994 John Wiley & Sons, Inc.