Factors affecting membrane fouling in filtration of Saccharomyces cerevisiae in an internal ceramic filter bioreactor

Filtration of ethanol fermentation medium and broth by using symmetric and asymmetric ceramic membranes has been studied in an internal filter bioreactor. Factors studied included membrane structure and pore size, medium sterilization, and concentrations of glucose, yeast extract in the medium, yeast cell and protein in broth. The aim was to determine the main factors responsible for the decline in filtration performance during ethanol fermentation by Saccharomyces cerevisiae. Flux index (Fi) of a new concept has been developed to evaluate the degree of flux decline during the membrane fouling process. Fi was defined as the ratio of the membrane flux at certain filtration time (t?=?t) to the initial (t?=??0) flux of pure water, not the initial (t?=?+0) flux of the test fluid. Flux with sterilized medium was approximately two-fold higher than that with unsterilized medium although the reason could not be explained clearly. Glucose, interaction between glucose and yeast extract, yeast cells, and proteins in fermentation broth were found to play an important part in membrane fouling. Fi of the symmetric membrane decreased to a less extent than that of the asymmetric membrane with increasing glucose concentration. But, the result with various yeast cell concentrations turned out to be contrary. Fouling was more serious for asymmetric membrane during the filtration of fermentation supernatant. This was thought to be due to different fouling mechanisms for the two types of membrane.     

Oligomeric Structure and Functional Characterization of the Urea Transporter from Actinobacillus pleuropneumoniae

Urea transporters (UTs) facilitate urea permeation across cell membranes in prokaryotes and eukaryotes. Bacteria use urea as a means to survive in acidic environments and/or as a nitrogen source. The UT from Actinobacillus pleuropneumoniae, ApUT, the pathogen that causes porcine pleurisy and pneumonia, was expressed in Escherichia coli and purified. Analysis of the recombinant protein using cross-linking and blue-native gel electrophoresis established that ApUT is a dimer in detergent solution. Purified protein was reconstituted into proteoliposomes and urea efflux was measured by stopped-flow fluorometry to determine the urea transport kinetics of ApUT. The measured urea flux was saturable, could be inhibited by phloretin, and was not affected by pH. Two-dimensional crystals of the biologically active ApUT show that it is also dimeric in a lipid membrane and provide the first structural information on a member of the UT family.     

Identification and biochemical characterization of a GDSL-motif carboxylester hydrolase from Carica papaya latex

An esterase (CpEst) showing high specific activities on tributyrin and short chain vinyl esters was obtained from Carica papaya latex after an extraction step with zwitterionic detergent and sonication, followed by gel filtration chromatography. Although the protein could not be purified to complete homogeneity due to its presence in high molecular mass aggregates, a major protein band with an apparent molecular mass of 41 kDa was obtained by SDS-PAGE. This material was digested with trypsin and the amino acid sequences of the tryptic peptides were determined by LC/ESI/MS/MS. These sequences were used to identify a partial cDNA (679 bp) from expressed sequence tags (ESTs) of C. papaya. Based upon EST sequences, a full-length gene was identified in the genome of C. papaya, with an open reading frame of 1029 bp encoding a protein of 343 amino acid residues, with a theoretical molecular mass of 38 kDa. From sequence analysis, CpEst was identified as a GDSL-motif carboxylester hydrolase belonging to the SGNH protein family and four potential N-glycosylation sites were identified. The putative catalytic triad was localised (Ser³⁵-Asp³⁰⁷-His³¹⁰) with the nucleophile serine being part of the GDSL-motif. A 3D-model of CpEst was built from known X-ray structures and sequence alignments and the catalytic triad was found to be exposed at the surface of the molecule, thus confirming the results of CpEst inhibition by tetrahydrolipstatin suggesting a direct accessibility of the inhibitor to the active site.     

Anaerobic digestion by a ceramic membrane enclosed reactor

A reactor with an external pressure type ceramic membrane module was designed to complete a compact system which brings about a reduction of permeability power requirements. The applicability of the reactor to methane fermentation was examined using heat-treated liquor obtained from sewage sludge. The experiments were carried out at 3 steps of CODcr-loading (Run 1; 4.53, Run 2; 7.98, Run 3; 15.4 kg CODcr/m³·d). The permeation flux stabilized at 0.06 in Run 1 and 0.11 m³/m²·d in Run 2, where the membrane surface was backwashed with N₂ gas. In Run 3, the membrane surface was additionally washed by circulating the gas yielded by the fermentation to increase the permeation flux, which stabilized at 0.2 m³/m²·d. The CODcr removal was 79.3 in Run 1, 83.2 in Run 2, and 80.2% in Run 3, respectively. In all cases, the BOD removal exceeded 95%, and there was no significant difference in the gas yield, which was 0.41 to 0.43 (l/g CODcr supplied) and 0.50 to 0.54 (l/g CODcr decomposed). Since the BOD could be greatly reduced even at 0.6 d of HRT and the microbiol cell concentration could be easily controlled, it seems possible to that the effciency of this reactor can be further improved.     

Isolation and characterization of trypsin inhibitors from cowpea (Vigna unguiculata)

The trypsin inhibitor fraction from cowpea (Vigna unguiculata) has been purified and characterized. Although the total trypsin inhibitor as purified by affinity chromatography on immobilised trypsin was shown to be heterogeneous by gel electrophoresis and isoelectric focusing as well as by function, it was relatively homogeneous in MW (ca 17 000) on gel filtration. The total trypsin inhibitor was divided into inhibitors active against trypsin only and active against trypsin and chymotrypsin by affinity chromatography on immobilised chymotrypsin. The ‘trypsin-only’ inhibitor was the major component of the total trypsin inhibitor. It was shown by isoelectric focusing and gel electrophoresis to contain several isoinhibitors. Determination of the combining weight of this inhibitor and investigation of the complexes formed with trypsin by gel filtration indicated the presence of two protease binding sites per inhibitor molecule. The chymotrypsin/trypsin inhibitor was also shown to be composed of several isoinhibitors. On the basis of gel electrophoresis and gel filtration in dissociating and non-dissociating media both inhibitors were considered to be dimeric molecules with the subunits linked by disulphide bonds; this implies that the ‘trypsin-only’ inhibitor has one binding site per subunit.     

Separation of furfural from monosaccharides by nanofiltration

Furfural, found in the lignocellulosic prehydrolyzates at high concentration, is a strong inhibitor of growth and ethanol fermentation of Saccharomyces cerevisiae. Removal of furfural and concentration of monosaccharides were investigated by using two commercial nanofiltraton (NF) membranes with synthetic glucose-xylose-furfural solution as model. The effects of main operating parameters such as feed pH, permeation flux, temperature and feed concentration on the rejections of the three solutes, were studied. Results showed that rejections of the three solutes decreased with increasing feed pH and temperature, and increased with increasing permeation flux for both membranes. The concentrations of the three solutes had interaction effect on the rejection of furfural by NF90 membrane and rejections of the three solutes by NF270 membrane. Furthermore, the effects of two filtration modes, concentration and diafiltration, on the separation of furfural from monosaccharides were also investigated. With the two commercial NF membranes, concentration and purification of monosaccharides in the model solution can be accomplished.     

Studies on the characterization of the Rho(D) antigen

The Rh_o(D) antigen of red cell membranes was solubilized using ethylenediamine tetraacetic acid (EDTA) and 2-mercaptoethanol. The solubilized antigen was partially separated from other solubilized membrane components using molecular filtration. The antigen was treated with various enzymes to learn some of the chemical characteristics. It was found that the activity of the antigen, as measured by hemagglutination inhibition, was not affected by bee venom phospholipase A, Clostridium welchii phospholipase C, calf-intestinal alkaline phosphatase, Vibrio cholerae neuraminidase, pig kidney leucine aminopeptidase, bovine pancreatic carboxypeptidase A, a pig pancreatic carboxypeptidase B. However, the proteolytic enzymes, pronase, trypsin, chymotrypsin and papain, did destroy Rh_o(D) activity as measured by hemagglutination inhibition. These results indicate that protein is an important part of the active determinant of the Rh_o(D) antigen. The experiments by other investigators have shown that lipid is important to maintain the Rh_o(D) activity in the intact membrane; lipid probably helps to maintain the structural conformation of the Rh_o(D) molecule in its natural environment. The solubilized Rh_o(D) molecules are apparently not dependent on lipid for their Rh_o(D) activity.     

Specific Binding of Protoporphyrin IX to a Membrane-Bound 63 Kilodalton Polypeptide in Cucumber Cotyledons Treated with Diphenyl Ether-Type Herbicides

Porphyrin accumulation in excised cucumber cotyledons (Cucumis sativus L.) treated with a N-phenylimide S-23142 (N-[4-chloro-2-fluoro-5-propargyloxyphenyl]-3,4,5,6- tetrahydrophthalimide) and a diphenylether acifluorfen-ethyl (ethyl-5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitro benzoic acid) was studied. Most of the accumulated porphyrins were found in the membrane fractions of 6,000g and 30,000g pellets, forming a complex with a membrane polypeptide. The complex was solubilized with 1% n-dodecyl β-d-maltoside and its molecular mass was estimated to be 63,000 and 66,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation high performance liquid chromatography (HPLC), respectively. The polypeptide also existed in untreated cotyledons but had little protoporphyrin IX. The complex was also formed in vitro by mixing the 30,000g pellets from untreated cotyledons and authentic protoporphyrin IX. However, protoporphyrin IX formed the complex specifically with the 63,000 dalton polypeptide and not with the other proteins both in vivo and in vitro. At least four fluorescent porphyrins, including protoporphyrin IX, were found in the acetone extract of the cotyledons by HPLC using a reversed phase column. Protoporphyrin IX was one of the two porphyrins that formed the complex. These results suggest that S-23142 and acifluorfenethyl enhance the accumulation of protoporphyrin IX, which forms the complex with the membrane protein.     

Air slugs entrapped cross-flow filtration of bacterial suspensions

A novel cross-flow technique for membrane filtration of bacterial cell suspensions was established. This is an air slugs entrapped cross-flow method in which air slugs were generated by introducing air into the cross-flow stream. As air slugs moved along with cross-flow, the disturbance of cell sublayer formation on membrane surface was enhanced. As a consequence, filtration flux was improved and stabilized. The effect of air slugs on improving filtration flux was more pronounced in filtering gram-negative Escherichia coli cell than grampositive Brevibacterium flavum cell. Moreover, air slug was about 50% more effective on reducing filtration resistance using ultrafiltration (UF) membrane of 300,000 molecular weight cutoff (MWCO) than microfiltration (MF) membrane of 0.2 mum. (c)1993 John Wiley & Sons, Inc.     

Transient and stationary operating conditions on performance of lactic acid bacteria crossflow microfiltration

A filtration rig equipped with a tubular alumina membrane was used to study the performance of crossflow microfiltration of Lactobacillus helveticus. Experiments were performed at constant permeation flux. High cell concentrations and fast transient conditions to the stationary J adversely affected permeability. Membrane fouling was due to a fast irreversible layer formation and to a reversible cell cake. This microbial deposit characteristics were dependent on the ratio permeation flux/wall shear stress, J/tau(w). Fouling was faster and more severe when J/tau(w) was greater than a critical value of 1.15 L(-1) . h(-1) . m(-2) . Pa(-1). The disordered structure of this cell cake seemed to lead to a macromolecule deposit between the cells which adversely affected the membrane permeability. (c) 1996 John Wiley & Sons, Inc.     

Heatable carbon nanotube composite membranes for sustainable recovery from biofouling

Membrane filtration is one of the most reliable methods for water treatment. However, wider application is limited due to biofouling caused by accumulation of microorganisms on the membrane surface. This report details a heatable carbon nanotube composite membrane with self-cleaning properties for sustainable recovery from biofouling. Microfiltration polycarbonate/carbon-nanotubes hybrid membranes were fabricated using drawable nanotubes that maintained the porosity and provided electrical conductivity to the membrane. Less than 25 V potential and 2–3 W power increase membrane temperature to 100°C in ~10 s. This temperature is above what most microbial life, bacteria and viruses can handle. When this membrane was employed, filtered Escherichia coli collected on its surface were successfully annihilated within 1 min. Ohmic heating of this membrane could be an effective solution to combat biofouling and complications associated with membrane-based filtration. This is a novel and highly desirable approach to combat biofouling, due to its simplicity and economic advantage.     

Antioxidant Activity of Purified Protein Hydrolysates from Northern Whiting Fish (Sillago sihama) Muscle

In the present study, northern whiting fish (Sillago sihama) muscle was hydrolyzed with gastrointestinal enzymes (pepsin, trypsin and α-chymotrypsin) separately and the resulted protein hydrolysates were tested for antioxidant activities using DPPH radical scavenging activity and reducing power assays. The protein hydrolysate obtained from trypsin exhibited highest antioxidant activity. Further, it was fractionated by consecutive chromatography using anion exchange and gel filtration chromatography; the separated fractions were collected and evaluated for antioxidant activity. The results showed that fraction 2 exhibited high chelating activity (73.15 % at 0.5 mg/mL) and best radical scavenging activity for DPPH radical (55.16 % at 0.5 mg/mL), ABTS radical (57.98 % at 50 μg/mL), superoxide radical (39.55 % at 200 μg/mL) and hydroxyl radical (51.33 % at 100 μg/mL). In addition, the active fraction showed strong antioxidant activity in the inhibition of linoleic acid autooxidation (60 % at 0.5 mg/mL) and also it exhibited significant protective effect on DNA damage caused by hydroxyl radicals. The size of the active fraction was found to be <360.2 Da using mass spectroscopy. These results demonstrate that muscle protein hydrolysate from northern whiting fish could be a best alternative to produce natural antioxidant peptides.     

Purification and Characterization of a Protein Cryoprotective for Vibrio cholerae Extracted from the Prawn Shell Surface

A substance cryoprotective for Vibrio cholerae on the prawn shell surface was purified by ammonium sulfate precipitation and gel filtration. It was a protein of 81 kDa and called cryoprotective protein (CPP). The cryoprotective activity of this protein for V. cholerae was sensitive to heat at 100 C and trypsin treatment. In the presence of Mg ion the protein can bind to the bacterial cell surface. V. cholerae can adhere to the shell surface of the prawn. The number of adhered bacteria was reduced by treating the shell with anti-CPP serum, heat or by trypsin. The presence of Mg ion promoted the adherence. These results suggest that the CPP could serve as an adherence site for V. cholerae on the shell surface.     

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.     

Se-mediated domain-domain communication in Band 3 of human erythrocytes

Na₂SeO₃ could affect the anion flux of Band 3 of inside-out erythrocyte membrane vesicles (IOVs). Such effect was believed to be based on the interaction of SH groups of Band 3 with Na₂SeO₃. This effect could be eliminated when the cytoplasmic domain of Band 3 was proteolytically removed by trypsin. This suggested that SH groups in the cytoplasmic domain were involved in such interaction. Measurement of the pH dependence of intrinsic fluorescence intensity provided evidence that conformational changes of Band 3 occurred as a consequence of interaction with selenite. KI quenching of intrinsic fluorescence of Band 3 could also show that there was a conformational change in the cytoplasmic domain of Band 3 after reaction with Na₂SeO₃. Such conformational change in turn could be transmitted to the membrane domain of Band 3 monitored by quenching of intrinsic fluorescence of Band 3 using hypocrellin B (HB) (a photosensitive pigment obtained from a parasitic fungus growing in Yunnan, China). It is suggested that the cytoplasmic domain of Band 3 is not necessary for its anion flux, but is essential for the regulation (e.g., by Se) of its active site located at the membrane domain, and hence, it may provide evidence of communication between the cytoplasmic domain and the membrane domain of Band 3.     

ANIONIC SITES OF HUMAN ERYTHROCYTE MEMBRANES : I. Effects of Trypsin, Phospholipase C, and pH on the Topography of Bound Positively Charged Colloidal Particles

The effects of pH, trypsin, and phospholipase C on the topographic distribution of acidic anionic residues on human erythrocytes was investigated using colloidal iron hydroxide labeling of mounted, fixed ghost membranes. After glutaraldehyde fixation at pH 6.5–7.5, the positively charged colloidal particles were bound to the membranes in small randomly distributed clusters. The clusters of anionic sites were reversibly aggregated by incubation at pH 5.5 before fixation at the same pH. These results correlate with the distribution of intramembranous particles found by Pinto da Silva (J. Cell Biol. 53:777), with the exception that the distribution of anionic sites on a majority of the fixed ghosts at pH 4.5 was aggregated instead of dispersed. The randomly distributed clusters could be nonreversibly aggregated by trypsin or phospholipase C treatment of intact ghosts before glutaraldehyde fixation. Previous glutaraldehyde fixation prevented trypsin and pH induced aggregation of the colloidal iron sites. Evidence that N-acetylneuraminic acid groups are the principal acidic residues binding colloidal iron was the elimination of greater than 85% of the colloidal iron labeling to neuraminidase-treated cell membranes. Colloidal iron binding N-acetylneuraminic acid residues may reside on membrane molecules such as glycophorin, a sialoglycoprotein which contains the majority of the N-acetylneuraminic acid found on the human erythrocyte membrane.     

A predictive aggregate transport model for microfiltration of combined macromolecular solutions and poly-disperse suspensions: testing model with transgenic goat milk

To meet the technical challenge of recovering human IgG fusion protein from transgenic whole goat milk at reasonable cost with high purity and yield, a predictive aggregate transport model for microfiltration has been developed (Baruah and Belfort, 2003). Here, to test the model's predictability of permeate flux and mass transport, a comprehensive series of experiments with varying wall shear rate, feed temperature, feed concentration, and module design are presented. A very good fit was obtained between the model predictions and measurements for a wide variety of experimental conditions. For microfiltration module design comparison, a linear hollow fiber module (representing current commercial technologies) gave lower permeation flux and higher yield than a helical hollow fiber module (representing the latest self-cleaning methodology). These results are easily explained with the model that is now being used to define operating conditions for maximizing performance. The procedure described by the model is generalizable and can be used to obtain optimal filtration performance for applications other than milk.     

Affinophoresis of trypsins with an anionic affinophore

Affinophoresis (Shimura, K. and Kasai, K. (1982) J. Biochem. 92, 1615–1622) is a newly devised electrophoretic separation technique for biomolecules, using an affinophore. The affinophore is a macromolecular polyelectrolyte bearing affinity ligands. It migrates rapidly in an electric field, and molecules which have affinity for the ligand are carried with it and separated from other molecules. An anionic affinophore for trypsin was synthesized. p-Aminobenzamidine, a competitive inhibitor of trypsin, was coupled to one-fifth of the car?yl groups of polyacrylyl-β-alanyl-β-alanine by the use of water-soluble carbodiimide and the residual car?yls were converted to sulfonate groups by coupling with aminomethanesulfonic acid. Affinophoresis was carried out in 1% agarose gel plates, and the protein bands were detected with Coomassie brilliant blue R250. Enhanced migrations of bovine and Streptomyces griseus trypsins towards the anode were observed with the anionic affinophore. The migrations of inactive forms prepared by active site modifications were scarcely affected. However, the affinophore was not effective for Streptomyces erythreus trypsin, an anionic trypsin, probably because of ionic repulsion between the anionic molecules. S. griseus trypsin was separated from Pronase by affinophoresis.     

Regulating the Skin Permeation Rate of Escitalopram by Ion-pair Formation with Organic Acids

In order to regulate the skin permeation rate (flux) of escitalopram (ESP), ion-pair strategy was used in our work. Five organic acids with different physicochemical properties, benzoic acid (BA), ibuprofen (IB), salicylic acid (SA), benzenesulfonic acid (BSA), and p-aminobenzoic acid (PABA), were employed as counter-ions to regulate the permeation rate of ESP across the rabbit abdominal skin in vitro. The interaction between ESP and organic acids was characterized by FTIR and ¹³C NMR spectroscopy. Results showed that all organic acids investigated in this study performed a controlling effect on ESP flux. To further analyze the factors concerned with the permeation capability of ESP-acid complex, a multiple linear regression model was used. It is concluded that the steady-state flux (J) of ESP-acid complexes had a positive correlation with log K _o/w (the n-octanol/water partition coefficient of ion-pair complex) and pK _a (the acidity of organic acid counter-ion), but a negative correlation with MW (the molecular weight of ion-pair complex). The logK _o/w of ion-pair complex is the primary one in all the factors that influence the skin permeation rate of ESP. The results demonstrated that organic acid with appropriate physicochemical properties can be considered as suitable candidate for the transdermal drug delivery of escitalopram.     

Properties of rat liver plasma membrane adenylate cyclase after chromatography on O-diethylaminoethyl-cellulose and agarose-hexane-GTP: Sensitivity of partially purified and purified enzyme to Lubrol-PX, 5′-guanylylimidodiphosphate,and glucagon

Partially purified rat liver plasma membranes were enriched to yield a more glucagon-sensitive membrane fraction which was solubilized with Lubrol-PX. The supernate obtained after centrifugation at 165,000g was subjected to O-diethylaminoethyl anion exchange chromatography. An adenylate cyclase fraction was eluted and purified further by chromatography on agarose-hexane-GTP. The enzyme adsorbed to the affinity resin and was eluted with 0.5 m Tris-HCl. The protein isolated by chromatography on the affinity resin was homogenous by conventional acrylamide gel electrophoresis; one band was observed in sodium dodecyl sulfate. The purified enzyme was free of nucleotide phosphohydrolases found in the parent solubilized membrane preparation. The anion exchange product was not sensitive to glucagon; Lubrol-PX and 5′-guanylylimidodiphosphate [Gpp(NH)p] decreased the activity of this fraction. In the presence of detergent or guanyl nucleotide, glucagon, at 10^?6m, increased enzyme activity by 30 and 21%, respectively, to a statistically significant degree, but not above basal levels. Adenylate cyclase was also purified by subjecting the 165,000g supernate directly to agarose-hexane-GTP; agarose-hexane-ATP or agarose-hexane was not effective. The affinity-derived material was associated with 85 nmol of Lubrol-PX/mg of protein. When calculated on the basis of a molecular weight of 150,000 for detergent-free protein after gel filtration on Bio-Gel A-0.5 m, there was 13 mol of detergent/mol of the enzyme obtained by chromatography on the affinity resin. The direct affinity product was insensitive to glucagon and Gpp(NH)p; enzyme activity varied as a function of Lubrol concentration.