Viability of an ectomycorrhizal fungus during cross-flow filtration

Factors affecting the viability and infectivity of an ectomycorrhizal fungus during moderate concentration by cross-flow filtration were determined. Mycelial suspensions were concentrated with three commercial membrane filters (Prostak Millipore Co., M14 Tech-Sep Co. and Ceraflo Norton Co.) under aseptic conditions. Medium components may reduce the filtration rate due to their low solubility. An antifoam agent did not reduce the average flux rates as much as did the malt extract. Clear unobstructed channels (I.D. 6mm) of the tubular modules (Tech-Sep) gave the best results both in terms of performance (filtration rate) and cell viability. Shear stresses caused by pumping and flow through narrow retentate channels were probably responsible for lowering viability and infectivity. There was no linear relationship between permeate fluxes and cell concentration. There is an optimum pore size both in terms of performance (filtration rate) and cell viability. Physical blockage of large pores by hyphae could explain lower permeate flux rates than those obtained with lower pore sizes membranes.     

Microfiltration of recombinant yeast cells using a rotating disk dynamic filtration system

To develop a highly efficient cell harvest step under time constraint, a novel rotating disk dynamic filtration system was studied on the laboratory scale (0.147-ft.(2) nylon membrane) for concentrating recombinant yeast cells containing an intracellular product. The existing cross-flow microfiltration method yielded pseudo-steady state flux values below 25 LMH (L/m(2). h) even at low membrane loadings (10 L/ft.(2)). By creating high shear rates (up to 120,000(-1)) on the membrane surface using a rotating solid disk, this dynamic filter has demonstrated dramatically improved performance, presumably due to minimal cake buildup and reduced membrane fouling. Among the many factors investigated, disk rotating speed, which determines shear rates and flow patterns, was found to be the most important adjustable parameter. Our experimental results have shown that the flux increases with disk rotating speed, increases with transmembrane pressure at higher cell concentrations, and can be sustained at high levels under constant flux mode. At a certain membrane loading level, there was a critical speed below which it behaved similarly to a flat sheet system with equivalent shear. Average flux greater than 200 LMH has been demonstrated at 37-L/ft.(2) loading at maximum speed to complete sixfold concentration and 15-volume diafiltration for less than 100 min. An order of magnitude improvement over the crossflow microfiltration control was projected for large scale production. This superior performance, however, would be achieved at the expense of additional power input and heat dissipation, especially when cell concentration reaches above 80 g dry cell weight (DCW)/L. Although a positive linear relationship between power input and dynamic flux at a certain concentration factor has been established, high cell density associated with high viscosity impacted adversely on effective average shear rates and, eventually, severe membrane fouling, rather than cake formation, would limit the performance of this novel system. (c) 1995 John Wiley & Sons, Inc.     

Influence of fermentation conditions and microfiltration processes on membrane fouling during recovery of glucuronane polysaccharides from fermentation broths.

We have investigated the recovery of exopolysaccharides produced by Sinorhizobium meliloti M5N1 CS bacteria from fermentation broths using different membrane filtration processes: cross-flow filtration with a 7 mm i.d. tubular ceramic membrane of 0.5-microm pores under fixed transmembrane pressure or fixed permeate flux and dynamic filtration with a 0.2 microm nylon membrane using a 16-cm rotating disc filter. With the tubular membrane, the polysaccharide mass flux was mainly limited by polymer transmission that decayed to 10% after 90 min. The mass flux of polymer produced under standard fermentation conditions (70 h at 30 degrees C) stabilized after 70 min to 15 g/h/m(2). This mass flux rises to 36 g/h/m(2) when the mean stirring speed during fermentation is increased and to 123 g/h/m(2) when fermentation is extended to 120 h. In both cases, the mean molecular weight of polysaccharides drops from 4.0 10(5) g/mol under standard conditions to 2.7 10(5) g/mol. A similar reduction in molecular weight was observed when the fermentation temperature was raised to 36 degrees C without benefit to the mass flux. These changes in fermentation conditions have little effect on stabilized permeate flux, but raise significantly the sieving coefficient, due probably to molecular weight reduction and the filamentous aspect of the polymer as observed from SEM photographs. The polymer-mass flux was also increased by reducing transmembrane pressure (TMP) and raising the shear rate by inserting a rod in the membrane lumen. Operation under fixed permeate flux instead of constant TMP inhibited fouling during the first 4 h, resulting in higher sieving coefficients and polymer mass fluxes. The most interesting results were obtained with dynamic filtration because it allows operation at high-shear rates and low TMP. Sieving coefficients remained between 90 and 100%. With a smooth disc, the polysaccharide mass flux remained close to 180 g/h/m(2) at 1500 rpm and cell concentrations from 1 to 3 g/L. When radial rods were glued to the disc to increase wall shear stress and turbulence, the mass flux rose to 275 g/h/m(2) at the same speed and cell concentration.     

Crossflow filtration of yeast broth cultivated in molasses

A broth of yeast cells cultivated in molasses was crossfiltered with a thin-channel module. The permeation flux gradually decreased at a constant cell concentration. The flux was much lower than that obtained for yeast broth cultivated in yeast extract, polypeptone, and dextrose (YPD) medium during the filtration. The flux did not depend on the membrane pore size (0.45 to 5 mum). The steady-state flux was one-twentieth that calculated for a cake filtration mode from the amount of cake per unit filtration area and the specific resistance of the cake measured in a dead-end filtration apparatus. The lower flux was due to small particles (most of which were less than 1 mum in diameter) in the molasses. The mehanism of crossflow filtration of broths of yeast cells cultivated in molasses was clarified by analysis of the change in flux with time and observations with scanning electron microscopy. At the initial stage of crossflow filtration the yeast cells and particles from the molasses were deposited on the membrane to form the molasses were deposited on the membrane to form a cake in a similar way to dead-end filtration. After the deposition of cells onto the membrane ceased, the fine particles from molasses formed a thin layer, which had higher resistance than the cake formed next to the membrane. The backwashing method was effective to increase the flux. The flux increased low when the pore size was 0.45 to 0.08 mum, but using larger pores of 3 to 5 mum it returned almost to the bases line. (c) 1994 John Wiley & Sons, Inc.     

Effect of Permeate Drag Force on the Development of a Biofouling Layer in a Pressure-Driven Membrane Separation System

The effect of permeate flux on the development of a biofouling layer on cross-flow separation membranes was studied by using a bench-scale system consisting of two replicate 100-molecular-weight-cutoff tubular ultrafiltration membrane modules, one that allowed flow of permeate and one that did not (control). The system was inoculated with Pseudomonas putida S-12 tagged with a red fluorescent protein and was operated using a laminar flow regimen under sterile conditions with a constant feed of diluted (1:75) Luria-Bertani medium. Biofilm development was studied by using field emission scanning electron microscopy and confocal scanning laser microscopy and was subsequently quantified by image analysis, as well as by determining live counts and by permeate flux monitoring. Biofilm development was highly enhanced in the presence of permeate flow, which resulted in the buildup of complex three-dimensional structures on the membrane. Bacterial transport toward the membrane by permeate drag was found to be a mechanism by which cross-flow filtration contributes to the buildup of a biofouling layer that was more dominant than transport of nutrients. Cellular viability was found to be not essential for transport and adhesion under cross-flow conditions, since the permeate drag overcame the effect of bacterial motility.     

Pressure effects in cross-flow microfiltration of suspensions of whole bacterial cells

The effect of Trans-Membrane Pressure (TMP) on permeate flux during cross-flow microfiltration of bacterial cell suspensions in tubular ceramic membranes is studied experimentally. Continuous filtration experiments with suspensions of whole bacterial cells (Mycobacterium M156) show a dramatic permeate flux decline with increasing TMP. During the very early stages of the filtration process, a linear relationship between permeate flux and TMP is observed, suggesting an initial surface sorption of cells on the membrane surface. At longer times, the permeate flux vs. TMP data exhibit a critical pressure beyond which the permeate flux declines with increasing trans-membrane pressure. This is interpreted in terms of the formation of a compressible cake, whose permeability can be described through the Carman-Kozeny equation.     

Protein fouling during constant-flux virus filtration: Mechanisms and modeling

As biomanufacturers consider the transition from batch to continuous processing, it will be necessary to re-examine the design and operating conditions for many downstream processes. For example, the integration of virus removal filtration in continuous biomanufacturing will likely require operation at low and constant filtrate flux instead of the high (constant) transmembrane pressures (TMPs) currently employed in traditional batch processing. The objective of this study was to examine the effect of low operating filtrate flux (5–100 L/m²/h) on protein fouling during normal flow filtration of human serum Immunoglobulin G (hIgG) through the Viresolve® Pro membrane, including a direct comparison of the fouling behavior during constant-flux and constant-pressure operation. The filter capacity, defined as the volumetric throughput of hIgG solution at which the TMP increased to 30 psi, showed a distinct minimum at intermediate filtrate flux (around 20–30 L/m²/h). The fouling data were well-described using a previously-developed mechanistic model based on sequential pore blockage and cake filtration, suitably modified for operation at constant flux. Simple analytical expressions for the pressure profiles were developed in the limits of very low and high filtrate flux, enabling rapid estimation of the filter performance and capacity. The model calculations highlight the importance of both the pressure-dependent rate of pore blockage and the compressibility of the protein cake to the fouling behavior. These results provide important insights into the overall impact of constant-flux operation on the protein fouling behavior and filter capacity during virus removal filtration using the Viresolve® Pro membrane.     

Filtration-based perfusion of hybridoma cultures in protein-free medium: Reduction of membrane fouling by medium supplementation with DNase I

In this study, a filtration-based perfusion process was developed for the production of monoclonal antibodies (IgM) by suspended hybridoma cells grown in protein-free medium. It was found that the use of protein-free medium for perfusion culture generated the formation of numerous visible suspended particles consisting of dead cells and cellular debris aggregated into fibrous material. Surprisingly high apparent viabilities were observed in such protein-free cultures. In addition, membrane fouling occurred more rapidly in protein-free medium than in conventional serum-supplemented medium. By the addition of deoxyribonuclease I (DNase I) to the protein-free medium, it was possible to prevent the formation of aggregates and to follow the evolution of the total cell population more accurately. Moreover, DNase I significantly reduced the fouling of filtration membranes, and that, for two different types of separation systems (cross-flow and vortex-flow filtration) and two different types of membranes (polycarbonate and hydrophilized polysultone). From these results, it is clear that the presence of DNA fragments liberated following cellular death is playing an important role in membrane fouling. Longevity of filtration membranes was found to be considerably greater using a vortex-flow filtration module than with a static plate-and-frame cross-flow filtration module. The use of vortex-flow filtration of conjuction with DNase I allowed maintenance of perfusion cultures for more than 1 month without membrane fouling or antibody retention and with a constant permeate IgM concentration of 250 mg/L. Hybridomacells appeared to gradually adapt to increasing rotational speed in the vortex-flow filtration module.     

Comparative testing of tangential microfiltration for microbial cultures

In an attempt to extend and intensify the productive periods of bioprocesses, a self-cleaning tangential filtration device was examined. Built into a special-design bioreactor, its cell retention was evaluated for continuous-flow operation with selected examples of bacteria (Escherichia coli), yeasts (Sacharomyces cerevisiae), and filamentous fungi (Aspergillus niger). Performance characteristics such as filtration rates and cell accumulation were assessed as a function of filter rotational speed, operating pressure, cultivation time, and microfilter type (i.e., membrane or porous metallic). The highest flux of cell-free filtrate for each culture type was achieved using a 0.45-micron membrane-covered microfilter. While the respective yeast (S. cerevisiae) and bacterial (E. coli) cell concentrations were enhanced by as much as 16- and 8-fold over the batch growth levels, the representative A. niger fungal cultivation was less satisfactory because of progressively declining filtration rates limited by hydraulically resistant layers of microbial surface growth quite resistant to in situ filter backflushing with gas. Maximum steady-state flux was independent of operating pressure, yet was enhanced at rotational speeds up to about 800 rpm. Higher speeds offered no further improvements. The overall fermentation process was limited by the moderate levels of attainable flux which restricted the feed and dilution rates. The maximum attainable stabilized fluxes were 26-40 L/m(2) x h.     

Crossflow filtration of baker's yeast with periodical stopping of permeation flow and bubbling

The periodical stopping of permeation flow was applied to increase the permeation flux in crossflow filtration of commercially available baker's yeast cell suspension. The permeation flux after 3 h filtration in the crossflow filtration increased to 8 x 10(-5) m(3) /m(2) s (290 L/m(2) h) from 2 x 10(-5) m(3)/m(2) s (72 L/m(2) h) by applying the periodical stopping of permeation. Introduction of air bubbles during the stopping period of permeation further increased the flux.(c) John Wiley & Sons, Inc.     

Purification of chondroitin precursor from Escherichia coli K4 fermentation broth using membrane processing

Recently the possibility of producing the capsular polysaccharide K4, a fructosylated chondroitin, in fed-batch experiments was assessed. In the present study, a novel downstream process to obtain chondroitin from Escherichia coli K4 fermentation broth was developed. The process is simple, scalable and economical. In particular, downstream procedures were optimized with a particular aim of purifying a product suitable for further chemical modifications, in an attempt to develop a biotechnological platform for chondroitin sulfate production. During process development, membrane devices (ultrafiltration/diafiltration) were exploited, selecting the right cassette cut-offs for different phases of purification. The operational conditions (cross-flow rate and transmembrane pressure) used for the process were determined on an ?KTA cross-flow instrument (GE Healthcare, USA), a lab-scale automatic tangential flow filtration system. In addition, parameters such as selectivity and throughput were calculated based on the analytical quantification of K4 and defructosylated K4, as well as the major contaminants. The complete downstream procedure yielded about 75% chondroitin with a purity higher than 90%.     

Purification during crossflow electromicrofiltration of fermentation broth

The present study was to investigate the purification of a fermentation broth by an electromicrofiltration membrane. Microfiltration runs with a crude and a centrifuged broth, with solution of particles recovered from centrifugation and with permeates from microfiltration experiments were thus compared.Microfiltration performances were governed by colloids and small particles that induced sharp initial flux declines. For these results, the evolution of the overall membrane resistance was increased by 80% in comparison with the electromicrofiltration membrane. The main focus of this study was set on the enhancement of the filtrate flux by an electric field. This pressure electrofiltration leads to a drastic improvement of the filtration by 100% and the filtration time was thereby reduced. Pressure electrofiltration serves as an interesting alternative to the cross-flow filtration and it effectively separates advantageous constituents such as amino acids and biopolymers from a fermentation broth. They were equally maintained during the microelectrofiltration, although they were significantly reduced by 45% by the microfiltration without the application of an electric field. Accordingly, since the electrofiltration membrane was provided more permeability, this study experimentally demonstrates that the permeability inside a membrane can be controlled using an electric field.     

Understanding and modeling alternating tangential flow filtration for perfusion cell culture

Alternating tangential flow (ATF) filtration has been used with success in the Biopharmaceutical industry as a lower shear technology for cell retention with perfusion cultures. The ATF system is different than tangential flow filtration; however, in that reverse flow is used once per cycle as a means to minimize fouling. Few studies have been reported in the literature that evaluates ATF and how key system variables affect the rate at which ATF filters foul. In this study, an experimental setup was devised that allowed for determination of the time it took for fouling to occur for given mammalian (PER.C6) cell culture cell densities and viabilities as permeate flow rate and antifoam concentration was varied. The experimental results indicate, in accordance with D'Arcy's law, that the average resistance to permeate flow (across a cycle of operation) increases as biological material deposits on the membrane. Scanning electron microscope images of the post‐run filtration surface indicated that both cells and antifoam micelles deposit on the membrane. A unique mathematical model, based on the assumption that fouling was due to pore blockage from the cells and micelles in combination, was devised that allowed for estimation of sticking factors for the cells and the micelles on the membrane. This model was then used to accurately predict the increase in transmembane pressure during constant flux operation for an ATF cartridge used for perfusion cell culture. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1291–1300, 2014     

Factors affecting the performance of crossflow filtration of yeast cell suspension

Factors affecting the performance of crossflow filtration were investigated with a thin-channel module and yeast cells. In crossflow filtration of Saccharomyces cerevisiae cells cultivated with YPD medium (Yeast extract, polypeptone, and dextrose) and suspended in saline, a steady state was attained within several minutes when the cell concentration was low and the circulation flow rate was high. The steady-state flux and the change in flux during the initial unsteady state were explained well by conventional filtration theory, with the amount of cake deposited and the mean specific resistance to the cake measured in a dead-end filtration apparatus used in calculation. When the circulation flow rate was lower than a critical value, a part of the channel of the crossflow filtration module was plugged with cell cake, and thus the steady-state flux was low. In crossflow filtration of suspensions of commercially available baker's yeast, the flux gradually decreased, and the flux after 8 h of filtration was lower than the value calculated by filtration theory. Fine particles contaminating the baker's yeast was responsible for the decrease. A similar phenomenon was responsible for the decrease. A similar phenomenon was observed in crossflow filtration of a broth of S. cerevisiae cells cultivated in molasses medium, which also contains such particles, had no effect of the permeation flux during crossflow filtration. (c) 1993 John Wiley & Sons, Inc.     

Performance of an anaerobic membrane bioreactor in which granular sludge and dynamic filtration are integrated

To alleviate the fouling of a filter, simple substrates, dynamic filtration, and granular sludge were applied in an anaerobic membrane bioreactor (AnMBR). The results showed that under a transmembrane pressure < 20 kPa, the filter flux ranged between 15 and 20 l (m^?2 h)^?1 for a period of 30 days. The flux was higher than the typical flux of AnMBRs with conventional membranes and most current dynamic filters. In addition, the low cost of the filter avoided the need for a higher flux. Moreover, a stable granular sludge bed, which consumed all volatile fatty acids, was maintained. A compact fouling/filtration layer formed on the filter, which contributed to low effluent chemical oxygen demand concentrations and turbidity. In addition, substrate scarcity in the filtration zone resulted in the evolution of diverse bacteria on the filter.     

Combined in-fermenter extraction and cross-flow microfiltration for improved inclusion body processing

In this study we demonstrate a new in-fermenter chemical extraction procedure that degrades the cell wall of Escherichia coli and releases inclusion bodies (IBs) into the fermentation medium. We then prove that cross-flow microfiltration can be used to remove 91% of soluble contaminants from the released IBs. The extraction protocol, based on a combination of Triton X-100, EDTA, and intracellular T7 lysozyme, effectively released most of the intracellular soluble content without solubilising the IBs. Cross-flow microfiltration using a 0.2 microm ceramic membrane successfully recovered the granulocyte macrophage-colony stimulating factor (GM-CSF) IBs with removal of 91% of the soluble contaminants and virtually no loss of IBs to the permeate. The filtration efficiency, in terms of both flux and transmission, was significantly enhanced by in-fermenter Benzonase digestion of nucleic acids following chemical extraction. Both the extraction and filtration methods exerted their efficacy directly on a crude fermentation broth, eliminating the need for cell recovery and resuspension in buffer. The processes demonstrated here can all be performed using just a fermenter and a single cross-flow filtration unit, demonstrating a high level of process intensification. Furthermore, there is considerable scope to also use the microfiltration system to subsequently solubilise the IBs, to separate the denatured protein from cell debris, and to refold the protein using diafiltration. In this way refolded protein can potentially be obtained, in a relatively pure state, using only two unit operations.     

Cross-flow membrane microfiltration of a bacteriol fermentation broth

Although cross-flow membrane filtration is a very attractive option for harvesting cells and recovering enzymes from cell homogenates, the process is not without its problems. Foremost of these is the deposit of dissolved and suspended solutes onto the membrane surface during operation. The formation of these dense and sometimes compressive sublayers (often called cakes) offers additional resistance to axial and permeate flows and often affects the retention characteristics of the process. In view of the complex nature of the sublayer formation process and its sensitivity to cross-flow velocity, this investigation was undertaken to determine the main factors responsible for the decline in performance during the harvesting of B. polymyxa broth by membrane microfiltration. System parameters varied include axial flow rate, concentration of cells, proteins and other components in the feed, membrane materials (ceramic, polypropylene, and stainless steel), and cleaning methods. To help explain the observed results, a new mass transport model-the solids flux model-based on the assumptions that back migration of particles from the sublayer or membrane surface is negligible and that particles that reach the solid-solution interface attach (stick) completely, is tested. Using a variety of diagnostic methods, magnesium ammonium phosphate precipitate is formed during steam sterilization of the medium and is implicated as the major foulant in this study.     

Separation of proteases from yellowfin tuna spleen by ultrafiltration

Separation of protease, trypsin and chymotrypsin from yellowfin tuna spleen extract by ultrafiltration (UF) using regenerated cellulose membranes with molecular weight cut off (MWCO) 30 and 100 kDa was studied. The 100 kDa membrane had a higher transmission of enzymes than that of the 30 kDa membrane. The enzyme transmission varied from 0.01 to 0.18 and from 0.6 to 0.8 for the 30 kDa membrane and 100 kDa membrane, respectively. The protein transmission was about 0.8 for both membranes. Increasing cross-flow rate and transmembrane pressure (TMP) increased permeate flux. The limiting fluxes at cross-flow rate 120, 240 and 360 L/h for the 30 kDa membrane were 17.3, 43.9 and 54.7 L/m2h, respectively and the limiting fluxes at the same flow rate for 100 kDa membrane were 34.1, 51.1 and 68.4 L/m2h, respectively. The separation of these proteases was achieved using the 30 kDa membrane. The purities of proteases were increased more than ten times at TMP 1.5 bar and cross-flow rate 360 L/h by diafiltration using 30 kDa membrane.     

Yeast suspension filtration: flux enhancement using an upward gas/liquid slug flow-application to continuous alcoholic fermentation with cell recycle

This study deals with the use of an upward gas/liquid slug flow to reduce tubular mineral membrane fouling. The injection of air into the feedstream is designed to create hydrodynamic conditions that destabilize the cake layer over the membrane surface inside the filtration module complex. Experimental study was carried out by filtering a biological suspension (yeast) through different tubular mineral membranes. The effects of operating parameters, including the nature of the membrane, liquid and gas flowrates, and transmembrane pressure, were examined. When external fouling was the main limiting phenomenon, flux enhancements of a factor of three could be achieved with gas sparging compared with single liquid phase crossflow filtration. The economic benefits of this unsteady technique have also been examined. To investigate the possibility of long-term operation of the two-phase flow principle, dense cell perfusion cultures of Saccharomyces cerevisiae were carried out in a fermentor coupled with an ultrafiltration module. The air injection allowed a high and stable flux to be maintained over 100 h of fermentation, with a final cell concentration of 150 g dry weight/L. At equal biomass level, a twofold gain in flux could be attained compared with classical steady crossflow filtration at half the cost.     

Role of membrane structure on the filtrate flux during monoclonal antibody filtration through virus retentive membranes

Virus removal filtration is a critical step in the manufacture of monoclonal antibody products, providing a robust size-based removal of both enveloped and non-enveloped viruses. Many monoclonal antibodies show very large reductions in filtrate flux during virus filtration, with the mechanisms governing this behavior and its dependence on the properties of the virus filter and antibody remaining largely unknown. Experiments were performed using the highly asymmetric Viresolve® Pro and the relatively homogeneous Pegasus™ SV4 virus filters using a highly purified monoclonal antibody. The filtrate flux for a 4 g/L antibody solution through the Viresolve® Pro decreased by about 10-fold when the filter was oriented with the skin side down but by more than 1000-fold when the asymmetric filter orientation was reversed and used with the skin side up. The very large flux decline observed with the skin side up could be eliminated by placing a large pore size prefilter directly on top of the virus filter; this improvement in filtrate flux was not seen when the prefilter was used inline or as a batch prefiltration step. The increase in flux due to the prefilter was not related to the removal of large protein aggregates or to an alteration in the extent of concentration polarization. Instead, the prefilter appears to transiently disrupt reversible associations of the antibodies caused by strong intermolecular attractions. These results provide important insights into the role of membrane morphology and antibody properties on the filtrate flux during virus filtration.