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.     

Enhanced system kLa and permeate flux with a ceramic membrane bioreactor

Summary A cross-flow ceramic membrane was coupled to a bioreactor to fulfil the alternate functions of process stream clarifier and primary aerator. At the same air supply rate (delivered as a back-flush), ceramic membranes provided up to 72% greater aeration than a ring-sparger located in the bioreactor. In Biol.ogical treatment of dairy food process waste, the air backflush had the additional benefit of inhibiting membrane fouling, thereby maintaining higher (by about 100%) permeate fluxes.     

Protein recovery from cell debris using rotary and tangential crossflow microfiltration

Protein recovery from a bacterial lysate was accomplished using microfiltration membranes in a flat crossflow filter and in a cylindrical rotary filter. Severe membrane fouling yielded relatively low long-term permeate flux values of 10(-4)-10(-3) cm/s (where I cm/s = 3.6 x 10(4) L/m(2) - h). The permeate flux was found to be nearly independent of transmembrane pressure and to increase with increasing shear rate and decreasing solids concentration. The flux increased with shear to approximately the one-third power or greater for the flat filter and the one-half power or greater for the rotary filter; the stronger dependence for the rotary filter is thought to result from Taylor vortices enhancing the back transport of debris carried to the membrane surface by the permeate flow. The average protein transmission or sieving coefficient was measured at approximately 0.6, but considerable scatter in the transmission data was observed. The largest sieving coefficients were obtained for dilute suspensions at high shear rate. The rotary filter provided higher fluxes than did the flat filter for dilute suspensions, but not for concentrated suspensions. (c) 1995 John Wiley & Sons, Inc.     

Production and separation of alpha-agarase from Altermonas agarlyticus strain GJ1B

This paper presents results on the production of alpha-agarase by a fermentation process and its separation using membrane microfiltration (MF). Optimization of fermentation conditions for alpha-agarase production using Altermonas agarlyticus grown on medium containing agar as a carbon source was done in batch, fed-batch and continuous fermentations. Continuous culture at a dilution rate of 0.03 h(-1) appeared to be best suited for production of alpha-agarase by this organism. At 0.03 h(-1) dilution rate, enzyme activity was 0.9 U/ml. Clarification of broth was done using a hollow-fibre microfiltration membrane. The influence of hydrodynamic parameters on permeate flux and enzyme activity was studied. The best performance was obtained with prefiltered fermentation broth. A stable permeate flux of about 250-270 ml/min.m2 and an enzyme retention rate between 0% and 25% was obtained at temperatures between 6 degrees C and 22 degrees C, transmembrane pressure of 100 mm Hg and fluid cross-flow velocity of 4 x 10(-2) m/s. From the experiments on concentration of fermentation broth, the best compromise between enzyme activity transmission and permeate flux was obtained at a concentration factor of 2.     

Lysozyme separation by hollow-fibre ultrafiltration

This paper discusses the purification of lysozyme from chicken egg white using hollow-fibre ultrafiltration (30kDa MWCO, polysulphone membrane). Lysozyme is preferentially transmitted through the membrane while the membrane largely retains other egg white proteins. Improvement in system hydrodynamics resulted in an increase in permeate flux while lysozyme transmission remained unaffected, leading to higher productivity. The percentage purity of lysozyme obtained was generally insensitive to system hydrodynamics. The permeate flux and productivity increased with increase in transmembrane pressure (TMP) before levelling off around 0.7bar. However, the TMP did not have any pronounced effect on the transmission and the purity of lysozyme. Experiments carried out in the diafiltration mode showed that moderately pure lysozyme (80-90%) could be obtained in an extended operation.     

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.     

Characterization of Bufo arenarum oocyte plasma membrane proteins that interact with sperm

Sperm-oocyte plasma membrane interaction is an essential step in fertilization. In amphibians, the molecules involved have not been identified. Our aim was to detect and characterize oocyte molecules with binding affinity for sperm. We isolated plasma membranes free from vitelline envelope and yolk proteins from surface-biotinylated Bufo arenarum oocytes. Using binding assays we detected a biotinylated 100 kDa plasma membrane protein that consistently bound to sperm. Chromatographic studies confirmed the 100 kDa protein and detected two additional oocyte molecules of 30 and 70 kDa with affinity for sperm. Competition studies with an integrin-interacting peptide and cross-reaction with an anti-HSP70 antibody suggested that the 100 and 70 kDa proteins are members of the integrin family and HSP70, respectively. MS/MS analysis suggested extra candidates for a role in this step of fertilization. In conclusion, we provide evidence for the involvement of several proteins, including integrins and HSP70, in B. arenarum sperm-oocyte plasma membrane interactions.     

Protein precipitate recovery using microporous membranes

The use of microporous membranes has been examined for the recovery of precipitated protein suspensions and related soluble protein. Membrane flux rates and soluble protein transmissions are reported for unstirred batch-cell studies and cross-flow experiments. The unstirred batch-cell gave soluble protein transmissions in the range 80-100% for feeds containing either soluble protein or a mix of soluble and isoelectrically precipitated protein. In all cases a sharp decline in flux was observed which was, for example, considerably greater for soluble protein at its isoelectric point, pH 4.6, than at pH 8.8. The presence of precipitated protein led to a further decrease in flux rate. In cross-flow studies, flux decline was eventually accompanied by a significant decline in soluble protein transmission. The flux protein-transmission characteristics of microporous membranes are discussed in terms of the interaction of the soluble and precipitated protein with the membrane.     

Topographical Studies on Subunit Polypeptides of the PS I Reaction Center Complex in the Thylakoid Membranes of the Thermophilic Cyanobacterium Synechococcus sp.

The permeability to protein molecules of the outer limitingmembranes and the thylakoid membranes in hypotonically shockedprotoplasts of the thermophilic cyanobacterium Synechococcussp. was studied by examining the effects of NaBr-washing andpronase E-digestion on phycobiliproteins and a 35 kDa proteinwhich are associated with the outer and inner surface of thethylakoid membranes, respectively, and by measuring photooxidationof added cytochrome c. All the results obtained indicate thatthe shocked protoplasts are in essence a homogenous right side-outthylakoid membrane preparation; the outer limiting membranesare leaky to protein molecules, whereas the thylakoid membranesare still impermeable to proteins. The thylakoid membranes becamepermeable to proteins when the protoplasts were mechanicallydisrupted. Following on from these findings, the membrane topology of subunitpolypeptides of the photosystem I reaction center complex wasstudied. Proteolytic digestion of shocked protoplasts with trypsinand pronase E indicated that four of the five subunit polypeptidesof the PS I reaction center complex are exposed at the stromalsurface of thylakoid membranes; two subunits of 14 and 13 kDawere selectively digested by trypsin, whereas two chlorophyll-bindingsubunits of 62 and 60 kDa were preferentially attacked by pronaseE. However, a 10 kDa subunit appears to be strongly resistantto the proteases. Experiments with mechanically disrupted protoplastsfailed to provide evidence for a uniform transmembrane organizationof the PS I subunits. (Received March 31, 1986; Accepted August 18, 1986)     

Crossflow microfiltration of recombinant Escherichia coli lysates after high pressure homogenization

Crossflow membrane filtration was used to process recombinant Escherichia coli cell lysates containing protein inclusion bodies after high pressure homogenization. The number of passes through the high pressure homogenizer changed the viscosities and average particle sizes of the cell lysates. The different cell lysates were processed with a hollow fiber unit containing microfiltration membranes and a plate and frame unit with either ultrafiltration or microfiltration membranes. There were differences in permeate flux and protein transmission for the various membranes with the best performing membranes giving permeate fluxes greater than 60 L m(-2) h(-1) and protein transmissions greater than 90%. For a given membrane, no differences were observed between the cell lysates following homogenization with one, two, and three passes at 83 MPa. The lack of a difference between the three lysates is due to their similarities with respect to the released macromolecules and the presence of small (<0.1 mum) cell debris. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 304-310, 1997.     

Microfiltration and ultrafiltration of polysaccharides produced by fermentation using a rotating disk dynamic filtration system

The recovery of exopolysaccharides (EPS) produced by Sinorhizobium meliloti bacteria by dynamic microfiltration was investigated using a rotating disk device designed in our laboratory, equipped with a 0.2 microm nylon membrane. This system differs from commercially available systems by the presence of vanes on the disk which produce a very important increase in permeate flux while yielding excellent EPS transmission. For polymers produced under standard fermentation conditions (70 h at 30 degrees C), the mass flux rose to 650 g h(-1) m(-2) using a disk equipped with 2 mm vanes rotating at 2000 rpm against 380 g h(-1) m(-2) with a smooth disk at the same speed. The maximum flux observed was 1560 g h(-1) m(-2) with a 6-mm vanes disk rotating at 3000 rpm and a 36 degrees C broth. An interesting finding was that the permeate flux J(f) for various disks can be correlated by the same function of the mean shear stress at the membrane tau(wm) according to J(f) = 4.6 tau(wm) (0.717) for a 30 degrees C broth, showing that the effect of vanes is merely to increase the shear stress by raising the fluid core velocity between the membrane and the disk. With 6-mm vanes the core angular velocity was found to be 84% of disk velocity vs. 45% for a smooth disk. When the fermentation temperature was increased to 36 degrees C to produce a lower molecular weight polymer, the permeate flux rose by about 250%, much more than what could be expected from the reduction in permeate viscosity and followed the same power law with membrane shear stress as for 30 degrees C. The same device was equipped with a PES 50 kDa membrane to concentrate EPS by ultrafiltration. Permeate fluxes were of the order of 160 L h(-1) m(-2) at 2000 rpm and 30 degrees C with nearly complete EPS rejection. Finally, the net electrical power consumed by the disk was measured by subtracting the power consumed without fluid from the power during filtration at the same speed. This power increases with speed and with the presence of vanes, but since the gain provided by the vanes is very high, the specific energy per m(3) of permeate is minimal with the highest vanes tested (6 mm) and maximal for smooth disks.     

Biological hydrogen production using a membrane bioreactor

A cross-flow membrane was coupled to a chemostat to create an anaerobic membrane bioreactor (MBR) for biological hydrogen production. The reactor was fed glucose (10,000 mg/L) and inoculated with a soil inoculum heat-treated to kill non-spore-forming methanogens. Hydrogen gas was consistently produced at a concentration of 57-60% in the headspace under all conditions. When operated in chemostat mode (no flow through the membrane) at a hydraulic retention time (HRT) of 3.3 h, 90% of the glucose was removed, producing 2200 mg/L of cells and 500 mL/h of biogas. When operated in MBR mode, the solids retention time (SRT) was increased to SRT = 12 h producing a solids concentration in the reactor of 5800 mg/L. This SRT increased the overall glucose utilization (98%), the biogas production rate (640 mL/h), and the conversion efficiency of glucose-to-hydrogen from 22% (no MBR) to 25% (based on a maximum of 4 mol-H(2)/mol-glucose). When the SRT was increased from 5 h to 48 h, glucose utilization (99%) and biomass concentrations (8,800 +/- 600 mg/L) both increased. However, the biogas production decreased (310 +/- 40 mL/h) and the glucose-to-hydrogen conversion efficiency decreased from 37 +/- 4% to 18 +/- 3%. Sustained permeate flows through the membrane were in the range of 57 to 60 L/m(2) h for three different membrane pore sizes (0.3, 0.5, and 0.8 microm). Most (93.7% to 99.3%) of the membrane resistance was due to internal fouling and the reversible cake resistance, and not the membrane itself. Regular backpulsing was essential for maintaining permeate flux through the membrane. Analysis of DNA sequences using ribosomal intergenic spacer analysis indicated bacteria were most closely related to members of Clostridiaceae and Flexibacteraceae, including Clostridium acidisoli CAC237756 (97%), Linmingia china AF481148 (97%), and Cytophaga sp. MDA2507 AF238333 (99%). No PCR amplification of 16s rRNA genes was obtained when archaea-specific primers were used.     

Detection of distinct endocytotic and phagocytotic activities in epithelial cells (pinacocytes) of freshwater sponges (Porifera, Spongillidae)

 Light and electron microscopical investigations using externally applied fluorescent and gold-labeled markers have revealed the existence of distinct endocytotic and phagocytotic activities in basal epithelial cells (pinacocytes) of the freshwater sponges Spongilla lacustris and Ephydatia es) of the f. The total rate of endocytotic membrane uptake, ascertained by the application of the cationic lipid probe TMA-DPH, was found to be 3.2% of the cell surface area/h. A typical fluid-phase endocytosis, demonstrated by the use of the water-soluble membrane-impermeable tracers BCECF-dextran and LY-CH, participates in endocytotic activity at a rate of 0.7% of the cell surface area/h and results in the formation of endosomes measuring 0.8–1 μm in diameter. Moreover, the application of labeled BSA succeeded in the detection of a receptor-mediated endocytosis amounting to a concentration-dependent uptake of 2.3–2.8% of the cell surface area/h. Coated pits and coated vesicles conveying the adsorbed BSA measure 0.3 μm in diameter and are covered on the cytoplasmic face with a clathrin-like protein (HC, 180 kDa; LC, 30 kDa). To facilitate phagocytotic activities, a series of fluorescent–labeled and chemically treated particles such as bacteria or latex beads have been successfully employed. Accordingly, the measured values of phagocytic membrane uptake between 1 and 8% of the cell surface area/h depend on the variety of size as well as the chemical nature of the different bioparticles and clearly point to phagocytosis as a key mechanism for providing freshwater sponges with nourishment. Accepted: 3 April 1997     

Influence of pH and salt concentration on the cross-flow microfiltration of BSA through a ceramic membrane

In this paper, the influence of pH in the 4–8 interval and NaCl concentration up to 25 mM on the cross-flow microfiltration of BSA was investigated. A tubular ceramic membrane with a pore size of 0.14 μm was employed and its point of zero charge was calculated. The evolution of permeate flow and BSA transmission with time was determined at 45 °C, a cross-flow velocity of 3.5 m/s and a transmembrane pressure of 100 kPa. The curves of permeate flow were explained according to the resistances in series model. Maximum protein transmission was obtained at the isoelectric point of BSA (4.9), with significant transmission also at the point of zero charge of the membrane and null transmission at pH 4 and 8. The highest permeate flow was observed at pH 7 and the lowest at 4.9. Finally, the addition of salt resulted to some extent in an improvement of both protein transmission and permeate flow.     

Cell harvesting by cross-flow microfiltration using a shear-enhanced module

Protein, produced by a bacterial culture of recombinant Vibrio cholerae, was separated from cells in a fermentation broth by cross-flow microfiltration. A new, mechanically agitated (rotational) shear filter, the DMF(TM) filter from Pall, was used to perform the separation. Higher protein recovery and permeate flux than commonly obtained during cell harvesting were demonstrated using sixfold concentration followed by twofold diafiltration. The transmembrane pressure only increased by 10 kPa when the flux was kept constant at 150 L/m(2) h during both concentration and diafiltration. The protein transmission was about 100% initially, and over 90% at the end of the concentration process. The protein transmission during the diafiltration was over 80%. The total recovery of protein was 97%. When using an enzymatic cleaning agent, no significant pure water flux decrease was detected during the course of the experiments. (c) 1996 John Wiley & Sons, Inc.     

Subcellular fractionation of pig stomach smooth muscle. A study of the distribution of the (Ca2+ + Mg2+)-ATPase activity in plasmalemma and endoplasmic reticulum

Isolated membrane vesicles from pig stomach smooth muscle (antral part) were subfractionated by a density gradient procedure modified in order to obtain an efficient extraction of extrinsic proteins. By using this method in combination with digitonin-treatment, an endoplasmic reticulum fraction contaminated with maximally 10 to 20% of plasma membranes was isolated, together with a plasma membrane fraction containing at most 30% endoplasmic reticulum. The endoplasmic reticulum and plasma membrane fractions differed in protein composition, reaction to digitonin, binding of wheat germ agglutinin, activities of marker enzymes and in the characteristics of the Ca2+ uptake. The Ca2+ uptake by the endoplasmic reticulum was much more stimulated by oxalate than the uptake by plasma membranes. Both fractions showed a (Ca2+ + Mg2+)-ATPase activity, but the largest amount of this enzyme was present in the plasma membranes. The study of the phosphorylated intermediates of the (Ca2+ + Mg2+)-ATPase by polyacrylamide gel electrophoresis revealed two phosphoproteins one of 130 kDa and one of 100 kDa (Wuytack, F., Raeymaekers, L., De Schutter, G. and Casteels, R. (1982) Biochim. Biophys. Acta 693, 45-52). The 130 kDa enzyme was predominant in the fraction enriched in plasma membrane whereas the distribution of the 100 kDa polypeptide correlated with the endoplasmic reticulum markers. The 130 kDa ATPase was the main 125I-calmodulin binding protein detected on nitrocellulose blots of proteins separated by gel electrophoresis. The (Ca2+ + Mg2+)-ATPase activity of the plasma membranes was higher than the (Na+ + K+)-ATPase activity, suggesting that the Ca2+ extrusion from these cells depends much more on the activity of the (Ca2+ + Mg2+)-ATPase than on Na+-Ca2+ exchange.     

Characteristics of the electrophysiological parameters of the embryonic cell membranes in the loach during Na+, K+-ATPase inhibition

Influence of ouabain on changes in transmembrane potential (TMP) and the membrane conductance has been studied in developing embryos of the loach Misgurnus fossilis L. Ouabain does not cause any significant changes in TMP level within 10-15 min after treatment but the membrane was then depolarized to a degree depending on developmental stage. Exposure to ouabain increases the conductance and changes the selectivity of membranes. Reversion potential of ionic current is then decreased from -70- -100 to -5- -30 mV. It is supposed that gradual membrane depolarization owing to the decrease of K+ gradient favors the membrane conductance changes under the influence of ouabain. It has been established that the active transport of Na+ and K+ takes part in realization of the rhythm of TMP level periodical oscillations during synchronous cleavage division.     

Forced-flow bioreactor for sucrose inversion using ceramic membrane activated by silanization

A forced-flow enzyme membrane reactor system for sucrose inversion was investigated using three ceramic membranes having different pore sizes. Invertase was immobilized chemically to the inner surface of a ceramic membrane activated by a silane-glutaraldehyde technique. With the cross-flow filtration of sucrose solution, the reaction rate was a function of the permeate flux, easily controlled by pressure. Using 0.5 mum support pore size of membrane, the volumetric productivity obtained was 10 times higher than that in a reported immobilized enzyme column reactor, with a short residence time of 5 s and 100% conversion of the sucrose inversion.     

Harvesting and concentration of human influenza A virus produced in serum-free mammalian cell culture for the production of vaccines

A process scheme for the harvesting and concentration of cell culture-derived human influenza A virus is presented. The scheme comprises two static filtration steps, chemical inactivation by beta-propiolactone and cross-flow ultrafiltration. Human influenza A virus A/PR/8/34 (H1N1) was produced in roller bottles with serum-free medium using MDCK cells as a host. Cultivations resulted in specific hemagglutination (HA) activities of 393 HAU (100 microL)(-1) and turbidities of 0.479 OD measured as the extinction of light at 700 nm (mean values are presented). The concentrations of soluble protein and DNA in the harvests were 72 microg/mL and 5.73 microg/mL, respectively. An average product yield of 79% based on HA activity was achieved after clarification by depth (85%) and microfiltration (93%). The turbidities of cell culture supernatants were reduced to 2% of their initial value. Concentration with 750 kDa hollow-fiber modules by a factor of 20 resulted in 97% recovery of the product when operated at a constant flux of 28 L/(m(2) h) and a wall shear rate of 9,500 s(-1). The amount of protein and DNA could be reduced to 16% and 33% of their initial amount, respectively. An overall product yield of 77% was achieved. Clarified supernatants and concentrates were further analyzed by non-reducing SDS-PAGE and agarose gel electrophoresis. Particle volume distributions of concentrates were obtained by dynamic light scattering analysis. From the results it can be concluded that the suggested process scheme is well suited for the harvesting and concentration of cell culture-derived influenza A virus.     

Physiological and biochemical analysis of L. tredecimguttatus venom collected by electrical stimulation

The L. tredecimguttatus venom was collected by electrical stimulation and systematically analyzed. Gel electrophoresis and RP-HPLC showed that the venom consisted primarily of proteins with molecular weights above 10 kDa, most of which were high-molecular-mass acidic proteins, with fewer proteins and peptides below 10 kDa. The most abundant proteins in the venom were concentrated at around 100 kDa, which included latrotoxins- the principal toxic components of the venom. Injection of the venom in mice and cockroaches P. americana gave rise to obvious poisoned symptoms, with LD50 values of 0.16 mg/kg and 1.87 microg/g, respectively. Electrophysiological experiments showed that the venom could block the neuromuscular transmission in isolated mouse phrenic nerve-hemidiaphragm and rat vas deferens preparations. The low-molecular-weight fraction (<10 kDa) of the venom had no effect on the transmission. Enzymatic analysis indicated that the venom possess activities of several kinds of hydrolases including hyaluronidase and proteases. These results demonstrated that L. tredecimguttatus venom was basically a large-protein-constituted venom and is one of the most poisonous spider venoms known in the world. The mammalian toxicity of the venom was based on its larger proteins rather than on smaller proteins and peptides, and its hydrolase activities might be involved in the latrodectism. The use of electrical stimulation method to collect the venom has the advantages of avoiding contamination and repeated use of the valuable L. tredecimguttatus venom resources.