Cell removal from fermentation broth by flocculation + sedimentation

Summary Cell separation by flocculation+sedimentation ofStreptoccocus equisimilis cultivation for hyaluronate lyase recovery, was investigated as a function of the pH of the fermentation broth, using three different cationic flocculants. The polyelectrolyte Superfloc N-100 appears to be the best of the three flocculants tested; after treatmen of pH 6.0 and 120 min free sedimentation, the cells are sedimented at 20% of the initial volume and 80% of the volume remained as a clear supernatant without loss of enzyme activity.     

Recovery of ammonium lactate and removal of hardness from fermentation broth by nanofiltration

Nanofiltration (NF) was investigated as an alternative to desalting electrodialysis (ED) and ion exchange for the recovery of ammonium lactate from fermentation broth. Three commercial NF membranes, NF45, NF70, and NTR-729HF, were characterized with 50 mM NaCl, MgSO(4), and glucose solutions. NF45 membrane was selected because it showed the lowest rejection of monovalent ion, the highest rejection of divalent ion, and the highest rejection of nonpolar molecule. Effects of the operating pressure were investigated in a range of 100-400 psig, on the flux, lactate recovery, and glucose and magnesium removal from a real fermentation broth containing about 1.0 M of ammonium lactate. The flux and recovery rate increased linearly with the pressure. However, lactate rejection also increased with the pressure, lowering the recovery yield. More magnesium ions and glucose were rejected as the pressure was increased, and at 400 psig, for example, magnesium ion was almost completely rejected, highlighting the chance of obviating the necessity of ion exchange to remove hardness, by using NF instead of desalting ED. Membrane fouling was not so severe as expected, considering the complex nature and a rather high concentration of the fermentation broth treated.     

Recovery of succinic acid from fermentation broth

Succinic acid is of high interest as bio-feedstock for the chemical industry. It is a precursor for a variety of many other chemicals, e.g. 1,4-butandiol, tetrahydrofuran, biodegradable polymers and fumaric acid. Besides optimized production strains and fermentation processes it is indispensable to develop cost-saving and energy-effective downstream processes to compete with the current petrochemical production process. Various methods such as precipitation, sorption and ion exchange, electrodialysis, and liquid–liquid extraction have been investigated for the recovery of succinic acid from fermentation broth and are reviewed critically here.     

Zeta potential as a measure of polyelectrolyte flocculation and the effect of polymer dosing conditions on cell removal from fermentation broth

Characterization of flocculation for cell removal from fermentation broth via polyelectrolyte addition is commonly based on qualitative methods such as physical appearance of the floc. The use of zeta potential as a quantitative measure of floc character was evaluated as an indicator of optimal polymer addition. Zeta potential was found to increase with increasing cationic polyelectrolyte dosage, but never reached zero regardless of the total amount of polymer added, indicating flocculation occurs at least partially through a bridging type mechanism. Experiments were conducted using various polymer concentrations (25-75 g/L) and dosing methods (batch, incremental and continuous addition) that resulted in variable overall polymer requirements to achieve optimum flocculation. Zeta potential was found to be constant at optimal floc character regardless of the total amount of polymer added, polymer concentration, or method of polymer addition. Experiments with two additional types of fermentation broth also showed characteristic zeta potentials at optimal flocculation. Polymer requirements to achieve a particular floc character can vary greatly, depending on polymer dosing conditions and fermentation batch. The effect of polymer dosing conditions on the polymer requirement to obtain optimal floc character was evaluated. Polymer dosing method and calcium concentration were both found to have a significant effect (P < 0.0001) with continuous polymer addition and high calcium concentration requiring less polymer than did batch polymer addition and low calcium concentration, respectively. Polymer dosing concentration did not significantly affect polymer requirement for optimal flocculation.     

Recovery of yeast invertase from ethanol fermentation broth

Summary Ethanol fermentation broth produced by an aggregated form ofSaccharomyces uvarum strain contained invertase when sucrose-based raw materials were used. The amount of invertase in the borth was in the range of 1.4 to 4.8 units/ml, which was affected by the dilution rate, the concentration of corn steep liquor, and the type of sugar used. The activity of invertase in the broth could be maintained at 0.8 units/ml over two months. When the broth was passed through DEAE-cellulose beads and eluted with a NaCl-Tris-HCl buffer solution, a 75% recovery yield of invertase with 9-fold purification and 30-fold concentration could be achieved.     

Extracellular enzyme loss during polyelectrolyte flocculation of cells from fermentation broth

Association of extracellular protein product with flocculated cells reduces product yield. Here, partitioning of the enzyme subtilisin between the liquid and polyelectrolyte-flocculated and sedimented Bacillus increased as the polymer dosage was increased beyond that necessary to obtain optimum floc character (brain floc) for cell removal by centrifugation. Partitioning to the cell floc is partly physical entrapment at all polymer dosages; however, at higher levels there is also direct interaction between the polyelectrolyte and enzyme. Enzyme loss was not likely due to pH denaturation during the flocculation process because conditions were within the stable pH range of the enzyme. The direct interaction between polyelectrolyte and enzyme was characterized through turbidimetric titrations and partitioning studies. Neither changes in the polymer feed concentration nor the method of polymer addition reduced the enzyme loss at dosages optimal for cell removal.     

LPS removal from an E. coli fermentation broth using aqueous two‐phase micellar system

In biotechnology, endotoxin (LPS) removal from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of bacterial expression systems widely used to manufacture therapeutic proteins. The viability of large‐scale industrial production of recombinant biomolecules of pharmaceutical interest significantly depends on the separation and purification techniques used. The aim of this work was to evaluate the use of aqueous two‐phase micellar system (ATPMS) for endotoxin removal from preparations containing recombinant proteins of pharmaceutical interest, such as green fluorescent protein (GFPuv). Partition assays were carried out initially using pure LPS, and afterwards in the presence of E. coli cell lysate. The ATPMS technology proved to be effective in GFPuv recovery, preferentially into the micelle‐poor phase (K_GFPuv < 1.00), and LPS removal into the micelle‐rich phase (%REM_LPS > 98.00%). Therefore, this system can be exploited as the first step for purification in biotechnology processes for removal of higher LPS concentrations. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Azocasein assay for alkaline protease in complex fermentation broth

Summary An azocasein assay has been developed for determination of alkaline protease in fermentation broth from a complex substrate containing ca. 50 g/l protein which to a high degree interferes with azocasein. Methods described in the literature have been found inaccurate as results deviate ca. 50% from true activity, so one of the methods is modified in order to eliminate interference. Proportionality between the relative azocasein concentration and the deviation from true activity is found. An azocasein concentration of 350 mg azocasein per ml sample gives satisfactory results with an accuracy of ±5%. Application of a standard addition method also improves the accuracy, but is laborious and less precise.Abbreviations a true enzyme activity - a _s activity determined from standard curve - AU Anson unit - c _A/T relative azocasein concentration (mg azocasein per mg total protein) - r ratio between measured and true enzyme activity - RD relative deviation from true value (%) - RSD relative standard deviation (%) - TCA trichloroacetic acid     

Recovery and purification of thuringiensin from the fermentation broth of Bacillus thuringiensis

Thuringiensin is a heat stable -exotoxin from Bacillus thuringiensis with a great potential for replacing the traditional chemical pesticides. A process using micellar-enhanced ultrafitration method to recover thuringiensin was significantly improved by the use of a spiral-wound membrane, which could be operated at a low transmembrane pressure drop. This method was performed by adding a surfactant cetylpyridinium chloride (CPC) into the fermentation broth. After the surfactant-thuringiensin conjugates were formed, the broth then passed through the ultrafiltration membrane and the retentate was collected. The results indicated the optimal concentration of CPC for producing a maximal recovery up to 99.3% is 4%. For purification, the centrifuged broth was further filtered by a membrane filter. The filtered solution then was mixed with 50% of activated carbon. The supernatant then was injected into a preparative HPLC. The eluate was collected during thuringiensin peak formation. This eluate was then concentrated by vacuum evaporation and dialysis using an electrodialyzer to remove the excess salts. The dialyzed solution was then crystallized by lyophilization. The purity of the thuringiensin crystal was identified by HPLC, capillary electrophoresis, and mass spectrometry.This revised version was published online in October 2005 with corrections to the Cover Date.     

Selective removal of ammonia from animal cell culture broth

Serum-free perfusion cultures of hybridoma TO-405 cells were carried out in spinner flasks coupled with zeolite A-3 packed beads. Ammonia was selectively removed from the culture broth by passing cell free permeate from ceramic cross flow filtration, through the zeolite packed bed. Ammonia concentration in the culture broth was effectively maintained between 1 to 4 mmol/l which was below the inhibitory concentration for cell growth. Maximum cell density levels of 10⁷ cells/ml as well as improved percentage cell viability higher than in serum-supplemented cultures were feasible in this system.The possible effects of shear stress, generated by variation of the flow rates of the broth through the ceramic filter module, on the growth of the hybridoma cells were investigated. Backwashing, by reversing the direction of the permeate, was found necessary to prolong the life of the filter. Variation of the flow rates of the broth through the ceramic module between 0.29 m/s to 0.59 m/s did not cause immediate cell damage but growth was repressed at the higher flow rate.This study also showed that glutamine appears to be one of the factors limiting the growth of the hybridoma cells.     

Recovery of penicillin G from fermentation broth with reactive extraction in a mixer-settler

Summary In a laboratory countercurrent mixer-settler, penicillin was recovered from its fermentation broth by extraction with Amberlite LA-2 in n-butylacetate at pH 5.0 and reextracted from the ion-pair complex containing a solvent phase with a buffer at 7.2–7.5 with an overall degree of extraction above 90 %.Symbols A amine - AHP complex - c concentration - C partition coefficent - E degree of extraction - HP penicillin acid - K_G equilibrium constant - P, P^– penicillin acid anion Indices aq aqueous phase - org organic phase - A amine - AHP complex - G overall - HP free acid - P penicillin     

Recovery of potassium clavulanate from fermentation broth by ion exchange chromatography and desalting electrodialysis

Ion exchange chromatography (IEC) and desalting electrodialysis (DSED) processes were developed for the recovery and purification of potassium clavulanate (KCA) from fermentation broth. A strong anion exchanger, Amberlite IRA 400 resin, a potassium acetate solution as equilibrium buffer, and a potassium chloride (KCl) solution as elution buffer were used for the recovery of KCA in IEC. In order to determine optimal operating conditions, the effects of various operating parameters such as equilibrium buffer pH and concentration, elution buffer concentration, gradient length, and volumetric flow rate on KCA recovery and by-product removal were investigated using a simulated fermentation broth. In the subsequent step of DSED, employing cation (Neocepta CMS, Tokuyama, Japan) and anion (Neocepta ACS, Tokuyama, Japan) exchange membranes were carried out to remove KCl that existed in a large amount in the ion exchanged solution. The effects of operation voltage and feed composition on the performance of DSED were investigated. Based on the operating conditions determined above, IEC and DSED were applied in sequence to an ultrafiltered fermentation broth. Almost complete removal of KCl was possible with no significant loss of KCA, although the KCA recovery was slightly lower than that with the simulated fermentation broth. Based on this observation, it was concluded that IEC and DESD could be an effective process combination for the recovery of KCA from fermentation broth.     

Addendum: Recovery of cellulases from spent broth of sugar beet pulp fermentation byTrichoderma reesei

Summary The exo- and endo-glucanases ofTrichoderma reesei were recovered after growth on sugar beet pulp by vacuum concentration followed by (NH₄)₂SO₄ or acetone precipitation. Final respective recoveries were 53% and 57% for the exoglucanase and 41% and 42% for the endoglucanase. The resuspended acetone precipitates of both enzymes showed no loss of activity after 30 d at 4°C, pH 4.8 and in the presence of 0.5% sorbic acid. (NH₄)₂SO₄-precipitated exoglucanase lost activity in the same period.

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Resumen Las exo y endoglucanasas deTrichoderma reesei se recuperaron después de su crecimiento en pulpa de remolacha azucarera mediante concentración al vacío seguida de precipitación con (NH₄)₂SO₄ o acetona. La recuperación final fue de 53% y 57% para la exoglucanasa y de 41% y 42% para la endoglucanasa. La resuspensión de los precipitados cetónicos de ambos enzimas no mostró perdidas de actividad después de 30 d a 4°C y pH 4.8 en presencia de ácido sórbico. La exoglucanasa precipitada con (NH₄)₂SO₄ perdió actividad durante este mismo periodo.

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Résumé Les exo- et endoglucanases deTrichoderma reesei ont été récupérées après croissance sur pulpe de betterave sucrière, par concentration sous vide après précipitation par le sulfate ammonique ou l'acétone. Les récupérations ont été respectivement de 53% et 57% pour l'exoglucanase et de 41% et 42% pour l'endoglucanase. Les resuspensions des deux enzymes après précipitation à l'acétone n'ont révélé aucune perte d'activité après 30 jours à 4°C, pH 4.8 et en présence de 0.5% d'acide sorbique. L'exoglucanase précipitée au sulfate ammonique avait perdu toute activité dans le même laps de temps.

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(Based on a paper presented at the First Latin American Congress on Biotechnology, Tucumán, Argentina, October 4–8, 1987)     

Separation of 2,3-butanediol from fermentation broth by reactive-extraction using acetaldehyde-cyclohexane system

Biochemical 2,3-butanediol is a renewable material with the potential to be used as an alternative fuel. However, in the lack of an effective separation process has limited its industrial application. In this paper, an effective process was achieved to separate 2,3-butanediol by reactive-extraction. Acetaldehyde and cyclohexane were chosen as the reactant and extractant, respectively. Ion-exchange resin HZ732 was used as the catalyst. Reaction equilibrium and a kinetic study on the reaction between 2,3-butanediol and acetaldehyde were investigated to provide basic data for process development. The reaction enthalpy and activation energy of reaction of 2,3-butanediol and acetaldehyde were ?30.05 ± 1.62 KJ/mol and 45.29 ± 2.89 KJ/mol, respectively. Feasible conditions were obtained as follows: operating temperature = 20°C, acetaldehyde: 2,3-butanediol = 0.5:1 (w/w), cyclohexane: fermentation broth = 0.5:1 (w/w), catalyst amount = 100 g/L, stirring rate = 500 rpm and three-stage counter-current extraction method was used. Under these conditions, the total yield rate of 2,3-butanediol from fermentation broth was over 90% and the mass fraction of 2,3-butanediol in the final product reached 99%.     

Desorption of carbon dioxide from fermentation broth

Rates of CO₂ desorption from fermentation broths under actual operating conditions were determined by measuring the CO₂ partial pressure in the exit gas. The concentrations of CO₂ physically dissolved in the broths were measured by the so-called tubing method. Values of k_La for CO₂ desorption calculated from these values agreed well with the k_La values for oxygen absorption corrected for the difference in gas diffusivities. The dissolved CO₂ concentration in the broth, which seems to bean important operating parameter, can easily be estimated from the CO₂ partial pressure in the exit gas, a more easily measurable quantity, if the k_La value is known. For a given value of k_La, assumption of perfect mixing or plug flow in the gas phase made little difference in the calculated values of the dissolved CO₂ concentration, indicating that the gas phase was probably in between perfect mixing and plug flow. In industrial fermentors, the CO₂ partial pressure in the exit gas can practically be assumed to be in equilibrium with the dissolved CO₂ concentration.     

Downstream processing of pullulan from fermentation broth

pullulan, a water soluble extracellular polysaccharide, was produced by downstream fermentation employing the strain Aureobasidium pullulans. To obtain pure biopolymer from the fermentation broth, it is necessary to harvest cells, heat the broth, remove the melanin pigments co-produced during fermentation, concentration, precipitate and dry. Centrifugation of the fermentation broth at 10,000 rpm for 15 min gave cell pellets that were discarded and a green–black supernatant containing melanin pigment was subjected to the heat treatment at 80 °C for 20 min in order to remove the protein in the fermentation broth. The supernatant was demelanized by oxidation with hydrogen peroxide, concentrated under vacuum, precipitated with ethanol and dried at 60 °C for 30 min. This procedure produced high purity pullulan that was comparable in color and texture to the commercial samples.     

Recovery of lactic acid from fermentation broth by the two-stage process of nanofiltration and water-splitting electrodialysis

A two-stage process of nanofiltration and water-splitting electrodialysis was investigated for lactic acid recovery from fermentation broth. In this process, sodium lactate is isolated from fermentation broth in the first stage of nanofiltration by using an NTR-729HF membrane, and then is converted to lactic acid in the second stage by water-splitting electrodialysis. To determine the optimal operating conditions for nanofiltration, the effects of pressure, lactate concentration, pH and known added impurities were studied. Lactate rejection was less than 5%, magnesium rejection approximated 45%, and calcium rejection was at 40%. In subsequent water-splitting electrodialysis, both the sodium lactate conversion to lactic acid and sodium hydroxide recovery, were about 95%, with a power requirement of 0.9∼1.0 kWh per kg of lactate.     

Recovery of ethanol from fermentation broths using selective sorption-desorption

Industrialized nations face a critical problem in replacing the sources of liquid fuels that traditionally have been supplied by petroleum. One solution that has gained increasing support in this country is the use of ethanol produced by fermentation of renewable biomass as an extender in, or supplement to, gasoline for transportation fuel. Distillation, the present method of separating ethanol from the fermentation broth, is an energy-intensive one and frequently uses more energy than is available from the ethanol recovered. There are many investigations under way to find alternative, less energy-intensive techniques for the ethanol-water separation. The separations method described in this article involves the use of solid materials to preferentially remove ethanol from fermentation broths. Subsequent stripping of the ethanol from the sorbent with a dry gas reduces dramatically the energy required for the separation. Three solid sorbents have been investigated experimentally. Their sorption/desorption characteristics are described, and their incorporation in an ethanol recovery process is evaluated. Three sorbents were investigated: two commercially available divinylbenzene crosslinked polystyrene resins in bead form (one with a nominal surface area of 300 m(2)/g, the other with 750 m(2)/g) and an experimental proprietary molecular sieve with hydrophobic properties. Equilibrium adsorption isotherms for two of the sorbents were obtained at ambient temperature (21 degrees C) for ethanol-water solutions containing up to 12 wt. % ethanol. In addition, 40 degrees C isotherms were obtained for the polystyrene sorbents. Although different, the equilibrium isotherms for the sorbents indicated that ethanol could be preferentially sorbed from a dilute solution. Column breakthrough curves indicated very favorable kinetics. Desorption of the ethanol was readily effected with warm (60-80 degrees C), dry nitrogen.     

Effects of solution environment on mammalian cell fermentation broth properties: Enhanced impurity removal and clarification performance

The processing of recombinant proteins from high cell density, high product titer cell cultures containing mammalian cells is commonly performed using tangential flow microfiltration (MF). However, the increased cellular debris present in these complex feed streams can prematurely foul the membrane, adversely impacting MF capacity and throughput. In addition, high cell density cell culture streams introduce elevated levels of process‐related impurities, which increase the burden on subsequent purification operations to remove these complex media components and impurities. To address this challenge, an evaluation of mammalian cell culture broth buffer properties was examined to determine if enhanced impurity removal and clarification performance could be achieved. A framework is presented here for establishing optimized mammalian cell culture buffer conditions, involving trade‐offs between product recovery and purification and improved clarification at manufacturing‐scale production. A reduction in cell culture broth pH to 4.7–5.0 induced flocculation and impurity precipitation which increased the average feed particle‐size. These conditions led to enhanced impurity removal and improved MF throughput and filter capacity for several mammalian systems. Feed conditions were further optimized by controlling ionic composition along with pH to improve product recovery from high cell density/high product titer cell cultures. Biotechnol. Bioeng. 2011; 108:50–58. © 2010 Wiley Periodicals, Inc.     

Analysis of broth rheology with cell morphology inCephalosporium fermentation

Summary Rheological properties inCephalosporium acremonium fermentation were quantitatively analysed. We obtained the correlation for consistency index (parameter of power law model) which could be estimated from the cell concentrations corresponding to morphological types. The correlated equation predicted the broth viscosity well.