Enzyme immobilization by fouling in ultrafiltration membranes: Impact of membrane configuration and type on flux behavior and biocatalytic conversion efficacy

Enzyme-immobilization in membranes accomplished by fostering membrane fouling was evaluated. Four different membrane configurations and five membranes were compared for immobilization of alcohol dehydrogenase (ADH) in terms of enzyme loading, permeate flux and final biocatalytic conversion. The membrane configuration impacted the efficiency of the enzyme-immobilization as well as the biocatalytic-membrane reaction, and the “sandwich mode”, with an extra polypropylene support above the membrane skin layer, worked best due to its high flux and stable conversion. Among the membranes, a GR51PP polysulphone membrane allowed for the highest flux during the reaction with the enzyme-immobilized membrane. At the same time, the lowest enzyme loading and low reaction stability were achieved for this membrane. Satisfactory enzyme loadings, stable conversions, but low flux rates were obtained for the PLTK and PLGC regenerated cellulose membranes. With these two highly hydrophilic membranes, the ADH enzyme activity was fully retained even after 24 h of storage of the membrane. Filtration blocking and resistance models were used to analyze the fouling/immobilization mechanisms and give explanations for the different results. The work confirms that fouling-induced enzyme immobilization is a promising option for enhancing biocatalytic productivity, and highlights the significance of the membrane type and configuration for optimal performance.     

Comparison of the performance of submerged membrane bioreactor (SMBR) and submerged membrane adsorption bioreactor (SMABR)

This study focuses on comparing the performance of submerged membrane bioreactor (SMBR) and submerged membrane adsorption bioreactor (SMABR) over a period of 20 days at a hydraulic retention time (HRT) of 3.1h. The effects of PAC on critical flux and membrane fouling were also investigated. The SMABR exhibited better results in terms of mixed liquor suspended solids (MLSS) growth, DOC removal (over 96%), COD removal (over 95%), transmembrane pressure (TMP) and oxygen uptake rate. Nearly 100% of bacteria and 100% of total coliforms were removed in both systems. The addition of PAC could maintain the critical flux at a lower TMP value (7.5 kPa), while irreversible fouling caused by PAC occurred when the filtration flux exceeded critical flux.     

A fluorescence-based indicator for nanofiltration fouling propensity caused by effluent organic matter (EfOM)

Effluent organic matter (EfOM) is one of the major foulants responsible for the occurrence of membrane fouling during advanced wastewater treatment using nanofiltration (NF) technology. In this present study, we have reported a simple fouling indicator based on the properties of fractional fluorescence and molecular weight, termed as fluorescence-size-index (FSI), to predict the fouling propensity of NF when filtrating EfOM. Specifically, EfOM collected from twenty-one real sewage samples were first analyzed to quantify their fluorescent compositions and concentrations. The results showed that the EfOM consisted mainly of humic-like substances, soluble organism metabolites and fulvic-like substances, characterized by small-molecule organic matters (<5 kDa) and hydrophobic fractions. Second, the major NF fouling fractions of EfOM were determined based on their fluorescent properties. It was observed that small-molecule hydrophobic components with humic-like fluorescence properties continuously influenced the flux decline rate throughout the whole operation, while macromolecular hydrophilic components with fluorescent properties of apparent aromatic hydrocarbon proteins were primarily responsible for the initial, rapid flux decline. Furthermore, the constructed FSI has proven to be useful in guiding the selection of pretreatment methods for preventing NF fouling.     

Effects of intermolecular thiol-disulfide interchange reactions on bsa fouling during microfiltration

Several studies have shown that one of the critical factors governing protein fouling of microfiltration membranes is the presence of denaturedand/or aggregated protein in the bulk solutions. Experiments were performed to evaluate the role of intermolecular disulfide interchange reactionson protein aggregation and membrane fouling during stirred cell microfiltration of bovine serum albumin (BSA). The flux decline during BSA filtration was quite dramatic due to the formation of a protein deposit thatfully covered the membrane pores. This flux decline could be completely eliminated by capping the free sulfhydryl group present on the BSA with eithera carboxymethyl or cysteinyl group, demonstrating the critical importance of this free thiol in the intermolecular aggregation reactions and, in turn, protein fouling. BSA aggregation during storage could be reduced by the addition of metal chelators (EDTA and citrate) or dithiothreitol, orby storage at lower pH (7.0) these solutions all had a significantly lower rate of fouling upon subsequent filtration. This behavior is completely consistent with the known chemistry of the thiol-disulfide interchange reaction, demonstrating that an understanding of these intermolecular (aggregation) reactions can provide a rational framework for the analysis and control of protein fouling in these membrane systems. (c) 1994 John Wiley & Sons, Inc.     

Surfactant pretreatment of a polysulfone ultrafilter for reduction of antifoam fouling

Effectiveness of surfactant precoat treatment of the polysulfone ultrafilter was first investigated for reduction of membrane fouling in ultrafiltration of antifoam. Fifteen different surfactants, including alcohols and synthetic nonionic surfactants, were tested. In general, pretreatment with nonionic surfactant gave a larger flux than that with alcohol did. The flux increase by pretreatment with nonionic surfactant depended on a hydrophile lipophile balance (HLB) value and type of hydrophobic tail. The most effective surfactant for reducing antifoam fouling among the 15 surfactants was Brij-58 which has an HLB value of 16 and a straight alkyl hydrophobic chain. The ultrafiltration flux of the membrane treated with Brij-58 was almost three times larger than that of untreated membrane. The precoat treatment with Brij-58 was the most effective for reducing antifoam fouling in terms of rejection properties.Furthermore, flux was also improved by the surfactant pretreatment in ultrafiltration of model process streams, such as fermentation media, broth, and yeast suspension with or without antifoam. The surfactant Brij-58 was found to be more effective for reducing membrane fouling in ultrafiltration of model stream YG compared with ethanol or Brij-35. The mean flux increase by the pretreatment with Brij-58 was about 80% in ultrafiltration of the model stream without antifoam. When antifoam was added to the model stream, flux was almost doubled by the pretreatment with Brij-58. The effectiveness of surfactant precoat treatment for reducing membrane fouling was also confirmed in terms of rejection properties. (c) 1994 John Wiley & Sons, Inc.     

Balance of Electrostatic and Hydrophobic Interactions in the Lysis of Model Membranes by E. coliα-Haemolysin

The relative weight of electrostatic interactions and hydrophobic forces in the process of membrane disruption caused by E. coliα-haemolysin (HlyA) has been studied with a purified protein preparation and a model system consisting of large unilamellar vesicles loaded with water-soluble fluorescent probes. Vesicles were prepared in buffers of different ionic strengths, or pHs, and the net surface charge of the bilayers was also modified by addition of negatively (e.g., phosphatidylinositol) or positively (e.g., stearylamine) charged lipids. The results can be interpreted in terms of a multiple equilibrium in which α-haemolysin may exist: aggregated HlyA ⇄ monomeric HlyA ⇄ membrane-bound HlyA. In these equilibria both electrostatic and hydrophobic forces are significant. Electrostatic forces become substantial under certain circumstances, e.g., membrane binding when bilayer and protein have opposite electric charges. Protein adsorption to the bilayer is more sensitive to electrostatic forces than membrane disruption itself. In the latter case, the irreversible nature of protein insertion may overcome electrostatic repulsions. Also of interest is the complex effect of pH on the degree of aggregation of an amphipathic toxin like α-haemolysin, since pH changes are not only influencing the net protein charge but may also be inducing protein conformational transitions shown by changes in the protein intrinsic fluorescence and in its susceptibility to protease digestion, that appear to regulate the presence of hydrophobic patches at the surface of the molecule, thus modifying the ability of the toxin to either aggregate or become inserted in membranes. Received: 29 October 1996/Revised: 4 February 1997     

Control of struvite precipitation by selective removal of $${\text{NH}}^{{\text{ + }}}_{{\text{4}}} $$ with dialyzer/zeolite in an anaerobic membrane bioreactor

This study focused on the mitigation of membrane fouling caused by struvite precipitation using a dialyzer/zeolite (D/Z) unit in an anaerobic membrane bioreactor. The D/Z unit was designed to selectively remove NH(4)+ ions, one of the main components of the inorganic foulant, struvite. The maximum mass transfer coefficient for NH(4)+ through the dialyzer was estimated to be 0.92 l m(-2)h(-1), whereas the Na-substituted zeolite had the highest ion exchange capacity with respect to ammonium among intact or differently pretreated zeolites. During a single passage of dialysate through the zeolite column, substantial NH(4)+ removal (in excess of 90%) was achieved, leading to the reduction in struvite precipitation in the digester. The D/Z unit played a significant role in controlling struvite precipitation, thereby enhancing permeate flux for the case of the ceramic membrane, in which struvite fouling would be more pronounced compared to the polymeric membrane. For the polymeric membrane, however, no significant improvement in flux was observed even with the D/Z unit because the fouling of the polymeric membrane was mainly due to the deposition of biomass rather than the struvite precipitation.     

A shortcut method for evaluation of protein deposition onto the membrane surface in crossflow ultrafiltration

In this study, a procedure for quantifying the surface deposition of proteins in crossflow ultrafiltration has been developed. The procedure consists of determining the protein adsorption behavior onto the membrane surface from a few dynamic measurements performed in a nonfiltration and a filtration mode, and evaluating the concentration polarization (CP) layer thickness based on the adsorption data. To predict the interdependence between the protein adsorption and CP, a simplified mathematical model has been formulated. The model was used to assess the protein adsorption and thus yield reduction in the ultrafiltration process at different protein concentration in the solution. As a case study, ultrafiltration of aqueous solutions of BSA and lysozyme (LYZ) was examined on a polyethersulfone membrane with the molecular weight cutoff of 10 or 100 kDa. The protein concentration in the solutions varied within a relatively low concentration range, i.e. below 10 mg mL^?1, characteristic for solvent exchange between sequential operations of protein purification by chromatography and extraction. Both proteins markedly differed in the mechanism of surface deposition; for BSA hydrophobic interactions were suggested to be dominant, whereas in case of LYZ electrostatic interactions contributed the most to the deposition mechanism. The effect of additives of the protein solutions, i.e. inorganic salts, PEG, and urea depended on the adsorption mechanism and was also specific for each protein. Nevertheless, the proposed procedure performed well in the evaluation of surface deposition and yield reduction, regardless of the protein type and its solvent environment.     

Implications of short and long term critical flux experiments for laboratory-scale MBR operations

This paper evaluates the critical flux obtained by different techniques including tests with different flux step lengths (20 and 40 min and 7 days) and modes of operation (continuous and intermittent) under low and high MLSS concentrations. The paper also analyses a couple of long-term tests (flow rate of 40 and 20 L/day) to obtain the time required to reach the critical flux experimentally and compares those values with the results obtained numerically from a mathematical model. It was found that intermittent mode with membrane relaxation was useful in controlling the fouling of membrane and in restoring the membrane from fouling at lower MLSS.     

A mechanistic study of in situ chemical cleaning-in-place for a PTFE flat sheet membrane: fouling mitigation and membrane characterization

This study aimed at unfolding the role and mechanisms of chemically enhanced cleaning-in-place (CIP) regimes in fouling control of polytetrafluoroethylene (PTFE) made flat sheet (FS) membrane bio-reactors (MBRs). The trans-membrane pressure (TMP) was successfully maintained below 10 kPa using a daily CIP regime consisting of 100 to 600 mg l^?1 of NaOCl and cake layer resistance control was shown to be critical for effective high-flux MBR operation. In contrast, in the control unit without the CIP, the TMP exceeded 35 kPa at a flux of 40 LMH. The extracellular polymeric substances associated with proteins (EPS_protein) were also controlled effectively with a daily application of the CIP to the fouled membrane. Moreover, the CIP prompted a thinner and looser bio-cake layer on the membrane surface, suggesting that in situ CIP can be a favorable method to control FS membrane fouling at high-flux MBR operation.     

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.     

Membrane fouling by cell-protein mixtures: in situ characterisation using multi-photon microscopy

Fouling of the membrane by cell and protein mixtures can result in severe flux declines, leading to the eventual need to clean or replace the membrane. In this study multi-photon microscopy, a fluorescence-based technique is used to 3-D image in situ the fouling of microfiltration membranes by suspensions containing combinations of washed yeast, bovine serum albumin (BSA) and ovalbumin. Appropriate fluorescent labelling allows the three foulant species to be clearly identified. Images correlate well with filtration data and clearly show the cake of yeast cells capturing protein aggregates. The proteins exhibited very different filtration behaviour. When filtering washed yeast together with ovalbumin and/or a 50:50 mixture by mass of BSA and ovalbumin, the ovalbumin fouling dominates the system. Capture of aggregates by the cake did not reduce fouling of the membrane by the protein and increased the resistance of the cake. For mixtures of BSA and washed yeast, the presence of a cake of yeast cells did reduce fouling of the membrane by the protein, however, the extra resistance due to the cake resulted in a flux lower than that when filtering BSA alone.     

Effect of membrane morphology on system capacity during normal flow microfiltration

Understanding the effects of membrane fouling on system capacity is critical for the successful design and scale-up of microfiltration systems. The underlying morphology and structure of the microfiltration membrane can have a significant effect on system capacity by altering the rate and extent of fouling. Experimental data were obtained for system capacity during protein microfiltration using several model membranes with both homogeneous and composite structures. Data were compared with predictions of a new model that can account for both pore blockage and cake formation, and also includes the effects of membrane morphology on internal flow profiles within the membrane. Membranes with highly interconnected pores have a significantly higher capacity due to the reduction in flux decline arising from the fluid flow under and around any surface blockage. The model calculations are in good agreement with the flux decline data, allowing far more accurate predictions of system capacity than for the commonly used V(max) analysis.     

Membrane binding of the colicin E1 channel: activity requires an electrostatic interaction of intermediate magnitude.

In vitro channel activity of the C-terminal colicin E1 channel polypeptide under conditions of variable electrostatic interaction with synthetic lipid membranes showed distinct maxima with respect to pH and membrane surface potential. The membrane binding energy was determined from fluorescence quenching of the intrinsic tryptophans of the channel polypeptide by liposomes containing N-trinitrophenyl-phosphatidylethanolamine. Maximum in vitro colicin channel activity correlated with an intermediate magnitude of the electrostatic interaction. For conditions associated with maximum activity (40% anionic lipid, I = 0.12 M, pH 4.0), the free energy of binding was delta G approximately -9 kcal/mol, with nonelectrostatic and electrostatic components, delta Gnel approximately -5 kcal/mol and delta Gel approximately -4 kcal/mol, and an effective binding charge of +7 at pH 4.0. Binding of the channel polypeptide to negative membranes at pH 8 is minimal, whereas initial binding at pH 4 followed by a shift to pH 8 causes only 3-10% reversal of binding, implying that it is kinetically trapped, probably by a hydrophobic interaction. It was inferred that membrane binding and insertion involves an initial electrostatic interaction responsible for concentration and binding to the membrane surface. This is followed by insertion into the bilayer driven by hydrophobic forces, which are countered in the case of excessive electrostatic binding.     

Probing protein colloidal behavior in membrane‐based separation processes using spectrofluorometric Rayleigh scattering data

One of the primary problems in membrane‐based protein separation is membrane fouling. In this study we explored the feasibility of employing Rayleigh light scattering data from fluorescence studies combined with chemometric techniques to determine whether a correlation could be established with membrane fouling phenomena. Membrane flux was measured in a dead‐end UF filtration system and the effect of protein solution properties on the flux decline was systematically investigated. A variety of proteins were used as a test case in this study. In parallel, the colloidal behavior of the protein solutions was assessed by employing multiwavelength Rayleigh scattering measurements. To assess the usefulness of Rayleigh scattering measurements for probing the colloidal behavior of proteins, a protein solution of β‐lactoglobulin was used as a base‐case scenario. The colloidal behavior of different β‐lactoglobulin solutions was inferred based on published data for this protein, under identical solution conditions, where techniques other than Rayleigh scattering had been used. Using this approach, good agreement was observed between scattering data and the colloidal behavior of this protein. To test the hypothesis that a high degree of aggregation will lead to increased membrane fouling, filtration data was used to find whether the Rayleigh scattering intensity correlated with permeate flux changes. It was found that for protein solutions which were stable and did not aggregate, fouling was reduced and these solutions exhibited reduced Rayleigh scattering. When the aggregation behavior of the solution was favored, significant flux declines occurred and were highly correlated with increased Rayleigh scattering. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Matrix-assisted laser desorption ionization mass spectrometry: a new tool for probing interactions between proteins and metal surfaces. Use in dental implantology.

The fixation in the bone of an artificial titanium tooth root is believed to be initiated by the rapid adsorption of the proteins present in the surgical cavity on the titanium surface. The study of this adsorption should make it possible to predict the osseointegration capacities of new implant surface treatments. We describe here a new method, based on matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS), for quantifying proteins adsorbed on titanium surfaces fully identical to these designed for implantology. The key step of this method is a new MALDI-MS sample preparation allowing the adsorbed proteins to be removed from the surface and to be homogeneously dispersed in the matrix crystals. The adsorption of a model protein (lysozyme) on two titanium surfaces (polished and sandblasted) was studied in order to evaluate the method. The absolute MALDI-MS intensity was shown to vary linearly with the amount of adsorbed lysozyme. After dipping the titanium surfaces for different times in lysozyme solutions at different concentrations, the maximum amount of adsorbed lysozyme was measured by MALDI-MS and was shown to correspond to a lysozyme monolayer, which is consistent with results described in the literature.     

Modification of beta-lactoglobulin by microbial transglutaminase under high hydrostatic pressure: localization of reactive glutamine residues

Microbial transglutaminase (mTG) mediated modification of bovine beta-lactoglobulin (bLG) at ambient and high hydrostatic pressure was investigated in order to characterize preferred sites of the crosslinking reaction by identifying reactive glutamine residues. bLG was labeled with triglycine (GGG) by incubation with mTG at ambient pressure or at 400 MPa, respectively, and was subjected to an enzymatic digestion with trypsin. The resulting peptides were separated and those containing glutamine residues modified with GGG were unambiguously identified using RP-HPLC with ESI-TOF-MS. For bLG treated with mTG at ambient pressure for 1 h at 40 degrees C, no labeling was observed, thus confirming that the native protein is no substrate for mTG. After incubation of the protein with mTG at 400 MPa for 1 h at 40 degrees C, four out of nine glutamine residues, namely at positions 5, 13, 35, and 59 were identified as accessible for the mTG catalyzed reaction, indicating partial unfolding of bLG under pressure and exposure of previously unaccesible glutamine residues. Thus, only a limited number of glutamine residues were substrates for mTG, which points to a pronounced substrate specificity of mTG toward individual glutamine residues within a protein.     

膜前助滤预处理对高藻水微滤膜污染的影响研究

研究采用添加硅藻土、植物棉、活性炭等3种不同预处理手段来过滤铜绿微囊藻,并考察未预处理及预处理后的藻液过滤过程中的过滤特性、有机物分布及膜污染特性。结果表明, 3种预处理手段对过滤通量均有所提高并减缓膜污染。其中,硅藻土预处理提高平均过滤通量达915%,明显优于其他助滤手段。活性炭预处理能够有效吸附芳香族蛋白质类荧光污染物,显著降低污染膜的不可逆化学污染阻力。通过OCT及SEM分析可知未预处理的高藻水直接过滤造成的膜污染最严重,饼层结构的粗糙度最低并且厚度也最小,而硅藻土通过优化饼层结构以达到缓解膜污染的效果。最后基于XDLVO理论结果也进一步证实硅藻土预处理手段对改善膜污染效果最好。研究结果对未来蓝藻水华膜处理技术的预处理手段研发具有指导意义。     

Statistical thermodynamics of membrane bending-mediated protein-protein attractions

Highly wedge-shaped integral membrane proteins, or membrane-adsorbed proteins can induce long-ranged deformations. The strain in the surrounding bilayer creates relatively long-ranged forces that contribute to interactions with nearby proteins. In contrast, to direct short-ranged interactions such as van der Waal's, hydrophobic, or electrostatic interactions, both local membrane Gaussian curvature and protein ellipticity can induce forces acting at distances of up to a few times their typical radii. These forces can be attractive or repulsive, depending on the proteins' shape, height, contact angle with the bilayer, and a pre-existing local membrane curvature. Although interaction energies are not pairwise additive, for sufficiently low protein density, thermodynamic properties depend only upon pair interactions. Here, we compute pair interaction potentials and entropic contributions to the two-dimensional osmotic pressure of a collection of noncircular proteins. For flat membranes, bending rigidities of approximately 100k(B)T, moderate ellipticities, and large contact angle proteins, we find thermally averaged attractive interactions of order k(B)T. These interactions may play an important role in the intermediate stages of protein aggregation. Numerous biological processes where membrane bending-mediated interactions may be relevant are cited, and possible experiments are discussed.