Effects of biofilm formation on membrane performance in submerged membrane bioreactors

The effects of biofilm formation on membrane performance were evaluated for a submerged membrane bioreactor (sMBR) system with six different types of micro- and ultrafiltration membranes (working volume = 19 l). After operation for 24 h the permeability of the membranes with a larger pore size (microfiltration) decreased to that of the membranes with a much smaller pore size (ultrafiltration). Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) confirmed that biofilms could reduce the influence of the membrane surface properties. The chemical oxygen demand (COD) removal efficiency was 95% for the oily wastewater treatment in the sMBR where the filtration process made an important contribution (47% based on feed COD). Significant enhancement in COD removal occurred at the initial filtration stage because of biofilm formation and the dynamic member role of the biofilm layer. Membranes with various pore sizes had approximately the same permeate quality that was attributed to the biofilm on the membrane surfaces. Nevertheless, the ultrafiltration membranes had 43% more COD removal efficiency than the other applied membranes at the beginning of filtration (before biofilm formation) because of the smaller pore sizes and better sieving.     

Anti-GA3-Me Antiserum with High Specificity toward the 13-Hydroxyl Group of C19-Gibberellins

Membrane clarification of green tea extract was studied as a treatment to reduce sediments in packaged drinks and as a pretreatment for concentration processes. The flux and variation of components were examined in dead-end and crossflow filtration with several types of membranes. In dead-end ultrafiltration, the flux reduction rate was small, although the initial flux was similar to the final flux in microfiltration. Prefiltration was effective in decreasing the reduction rate of flux. As the pore size of microfiltration membranes became smaller, the dry weight decreased gradually and the optical transmission at 660 nm increased. By ultrafiltration, 30–50% pectin, 3–11% catechins and, 7–20% caffeine were rejected. Crossflow filtration was effective in keeping the flux high. The ultrafiltration spiral membrane (pore size: 0.008 μm) was selected for repeated batch clarification of prefiltered green tea crude extract and showed reproducible performance.     

Removal of endotoxin from water by microfiltration through a microporous polyethylene hollow-fiber membrane

The microporous polyethylene hollow-fiber membrane has a unique microfibrile structure throughout its depth and has been found to possess the functions of filtration and adsorption of endotoxin in water. The membrane has a maximum pore diameter of approximately 0.04 micron, a diameter which is within the range of microfiltration. Approximately 10 and 20% of the endotoxin in tap water and subterranean water, respectively, was smaller than 0.025 micron. Endotoxin in these water sources was efficiently removed by the microporous polyethylene hollow-fiber membrane. Escherichia coli O113 culture broth contained 26.4% of endotoxin smaller than 0.025 micron which was also removed. Endotoxin was leaked into the filtrate only when endotoxin samples were successively passed through the membrane. These results indicate that endotoxin smaller than the pore size of the membrane was adsorbed and then leaked into the filtrate because of a reduction in binding sites. Dissociation of 3H-labeled endotoxin from the membrane was performed, resulting in the removal of endotoxin associated with the membrane by alcoholic alkali at 78% efficiency.     

Removal of endotoxin from water by microfiltration through a microporous polyethylene hollow-fiber membrane.

The microporous polyethylene hollow-fiber membrane has a unique microfibrile structure throughout its depth and has been found to possess the functions of filtration and adsorption of endotoxin in water. The membrane has a maximum pore diameter of approximately 0.04 micron, a diameter which is within the range of microfiltration. Approximately 10 and 20% of the endotoxin in tap water and subterranean water, respectively, was smaller than 0.025 micron. Endotoxin in these water sources was efficiently removed by the microporous polyethylene hollow-fiber membrane. Escherichia coli O113 culture broth contained 26.4% of endotoxin smaller than 0.025 micron which was also removed. Endotoxin was leaked into the filtrate only when endotoxin samples were successively passed through the membrane. These results indicate that endotoxin smaller than the pore size of the membrane was adsorbed and then leaked into the filtrate because of a reduction in binding sites. Dissociation of 3H-labeled endotoxin from the membrane was performed, resulting in the removal of endotoxin associated with the membrane by alcoholic alkali at 78% efficiency.     

Recovery of alkaline proteases by membrane filtration

This work describes the recovery of an extracellular alkaline protease from fermentation broths of a Bacillus sp ATCC 21536, at pH=10.0 using ultrafiltration (MWCO 100,000) and microfiltration (0.1 m) membranes in hollow fiber devices. The influence of membrane pore size and polymeric material and membrane filtration performance was studied. High protein recoveries and high average flux rates were obtained with polysulfone membranes. A decrease of concentration polarization was obtained, simultaneously with enhancement of filtration flux rate and enzyme recovery by using submicron sized charged particles. These polymers lead to flocculation and adsorption of whole cells and soluble factors from the fermentation broth. The best results were obtaiend by combination of cationic (0.1%) and anionic (0.04%) polymers.     

Analysis of protein fouling during ultrafiltration using a two-layer membrane model

Protein fouling can significantly alter both the flux and retention characteristics of ultrafiltration membranes. There has, however, been considerable controversy over the nature of this fouling layer. In this study, hydraulic permeability and dextran sieving data were obtained both before and after albumin adsorption and/or filtration using polyethersulfone ultrafiltration membranes. The dextran molecular weight distributions were analyzed by gel permeation chromatography to evaluate the sieving characteristics over a broad range of solute size. Protein fouling caused a significant reduction in the dextran sieving coefficients, with very different effects seen for the diffusive and convective contributions to dextran transport. The changes in dextran sieving coefficients and diffusive permeabilities were analyzed using a two-layer membrane model in which a distinct protein layer is assumed to form on the upstream surface of the membrane. The data suggest that the protein layer formed during filtration was more tightly packed than that formed by simple static adsorption. Hydrodynamic calculations indicated that the pore size of the protein layer remained relatively constant throughout the adsorption or filtration, but the thickness of this layer increased with increasing exposure time. These results provide important insights into the nature of protein fouling during ultrafiltration and its effects on membrane transport.     

Selective isolation of bacteria for metagenomic analysis: Impact of membrane characteristics on bacterial filterability

For indirect DNA extraction for metagenomics studies, bacterial cells can be effectively separated from sample debris by using a simple size exclusion technique, such as filtration, and thereafter lysed. The requirement for the optimal recovery of cells in filtrates is critical to achieve sufficient DNA yield and a representative population. Particles smaller than the filter pore size are expected to be found in the filtrate, whereas particles larger than the filter pore sizes are excluded. However, this is not always the case. It is established that the membrane pore size influences filtration efficiency to some degree. In addition the physicochemical characteristics of the filter suspension and characteristics of the microbial cells being filtered influence the exclusion property of a membrane. This review provides an overview of membrane filtration techniques and the factors that affect filterability of bacteria cells through a filter membrane. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:853–866, 2015     

Virus harvesting in perfusion culture: Choosing the right type of hollow fiber membrane

The use of bioreactors coupled to membrane-based perfusion systems enables very high cell and product concentrations in vaccine and viral vector manufacturing. Many virus particles, however, are not stable and either lose their infectivity or physically degrade resulting in significant product losses if not harvested continuously. Even hollow fiber membranes with a nominal pore size of 0.2 µm can retain much smaller virions within a bioreactor. Here, we report on a systematic study to characterize structural and physicochemical membrane properties with respect to filter fouling and harvesting of yellow fever virus (YFV; ~50 nm). In tangential flow filtration perfusion experiments, we observed that YFV retention was only marginally determined by nominal but by effective pore sizes depending on filter fouling. Evaluation of scanning electron microscope images indicated that filter fouling can be reduced significantly by choosing membranes with (i) a flat inner surface (low boundary layer thickness), (ii) a smooth material structure (reduced deposition), (iii) a high porosity (high transmembrane flux), (iv) a distinct pore size distribution (well-defined pore selectivity), and (v) an increased fiber wall thickness (larger effective surface area). Lowest filter fouling was observed with polysulfone (PS) membranes. While the use of a small-pore PS membrane (0.08 µm) allowed to fully retain YFV within the bioreactor, continuous product harvesting was achieved with the large-pore PS membrane (0.34 µm). Due to the low protein rejection of the latter, this membrane type could also be of interest for other applications, that is, recombinant protein production in perfusion cultures.     

Anti-biofouling property of vanillin on Aeromonas hydrophila initial biofilm on various membrane surfaces

Biofouling is a serious problem on filter membranes of water purification systems due to formation of bacterial biofilms, which can be detrimental to the membrane performance. Biofouling occurs on membrane surface and therefore greatly influences the physical and chemical aspects of the surface. Several membranes including microfiltration, ultrafiltration, and reverse osmosis (RO) membranes were used to learn about the anti-biofouling properties of vanillin affecting the membrane performances. Vanillin has been recognized as a potential quorum quenching compound for Aeromonas hydrophila biofilms. The initial attachment and dynamics of biofilm growth were monitored using scanning electron microscopy and confocal laser scanning microscopy. Biofilm quantities were measured using a plate count method and total protein determinations. Vanillin addition was effective in the prevention of biofilm formation on the tested membrane surfaces. Among the membranes, RO membranes made with cellulose acetate showed the most substantial reduction of biofilm formation by addition of vanillin. The biofilm reduction was confirmed by the results of surface coverage, biomass and protein accumulation. The HPLC spectrum of the spent culture with vanillin addition showed that vanillin may interfere with quorum sensing molecules and thus prevent the formation of the biofilms.     

Removal versus fragmentation of amyloid-forming precursors via membrane filtration

The presence of even minute amounts of protein aggregates in solution can significantly alter the kinetics of amyloid formation. Removal of such pre-existing aggregates is critical for reproducible analysis of amyloid formation. Here we examine the effects of membrane filtration on insulin fibrillization. We find that filtration of insulin with large pore membranes (≥ 100 nm) generally slows fibril formation relative to unfiltered solutions by removing pre-aggregated protein. Unexpectedly, filtration with small pore membranes (< 100 nm) showed no beneficial effect and, in some cases, accelerated insulin fibril formation. This effect may be due to fragmentation of pre-existing aggregates during filtration through small pore membranes, which can increase the number of amyloid-forming precursors. These findings reveal the complexity of removing protein aggregates via filtration and suggest optimal filtration protocols for conducting fibril formation analysis of insulin and similar amyloidogenic proteins.     

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.     

A forced-flow membrane reactor for transfructosylation using ceramic membrane

A forced-flow membrane reactor system for transfructosylation was investigated using several ceramic membranes having different pore sizes. beta-Fructofuranosidase from Aspergillus niger ATCC 20611 was immobilized chemically to the inner surface of a ceramic membrane activated by a silane-coupling reagent. Sucrose solution was forced through the ceramic membrane by crossflow filtration while transfructosylation took place. The saccharide composition of the product, which was a mixture of fructooligosaccharides (FOS), was a function of the permeate flux, which was easily controlled by pressure. Using 0.2 micrometer pore size of symmetric ceramic membrane, the volumetric productivity obtained was 3.87 kg m(-3) s(-1), which was 560 times higher than that in a reported batch system, with a short residence time of 11 s. The half-life of the immobilized enzyme in the membrane was estimated to be 35 days by a long-term operation.     

Adherence of bacteria, yeast, blood cells, and latex spheres to large-porosity membrane filters.

Strong adherence of bacteria, yeast, erythrocytes, leukocytes, platelets, spores, and polystyrene spheres to membrane filter materials was noted during filtration through membranes with pore size diameters much larger than the particles themselves. Quantitative recovery on the membrane filters of these particles from low-concentration suspensions was achieved during gravity- or vacuum-assisted filtration through membranes with pore diameters as much as 30 times that of the filtered particles. Mechanical sieving was not responsible. The phenomenon was judged to be electrostatic. It could be partially blocked by pretreating the filter with a nonionic surfactant (Tween 20), and elution of adherent particles was achieved with 0.05% Tween 20. Gram-positive cocci were removed from suspension more efficiently than gram-negative rods. The commonly used cellulose membranes adsorbed more bacteria, blood cells, and other particles than did polycarbonate filters. Of lesser adsorptive capacity were vinyl acetate, nylon, acrylic, and Teflon membranes. Backwashing with saline, serum, 6% NaCl, dextran solutions, or phosphate buffers of varying molality and pH removed only a fraction of adherent particles. Tween 20 (0.05%) eluted up to 45% of adherent particles in a single back-filtration. Selected filters quantitatively removed the particles tested, which then could be washed and subjected to reagents for a variety of purposes. It is important to anticipate the removal of particles during membrane filtration, since it is not a simple mechanical event.     

Hybrid process of BAC and sMBR for treating polluted raw water

The hybrid process of biological activated carbon (BAC) and submerged membrane bioreactor (sMBR) was evaluated for the drinking water treatment from polluted raw water, with the respective hydraulic retention time of 0.5 h. The results confirmed the synergetic effects between the BAC and the subsequent sMBR. A moderate amount of ammonium (54.5%) was decreased in the BAC; while the total removal efficiency was increased to 89.8% after the further treatment by the sMBR. In the hybrid process, adsorption of granular activated carbon (in BAC), two stages of biodegradation (in BAC and sMBR), and separation by the membrane (in sMBR) jointly contributed to the removal of organic matter. As a result, the hybrid process managed to eliminate influent DOC, UV₂₅₄, COD_Mn, TOC, BDOC and AOC by 26.3%, 29.9%, 22.8%, 27.8%, 57.2% and 49.3%, respectively. Due to the pre-treatment effect of BAC, the membrane fouling in the downstream sMBR was substantially mitigated.     

Experimental study of the anaerobic urban wastewater treatment in a submerged hollow-fibre membrane bioreactor at pilot scale

The aim of this study was to assess the effect of several operational variables on both biological and separation process performance in a submerged anaerobic membrane bioreactor pilot plant that treats urban wastewater. The pilot plant is equipped with two industrial hollow-fibre ultrafiltration membrane modules (PURON? Koch Membrane Systems, 30 m2 of filtration surface each). It was operated under mesophilic conditions (at 33 °C), 70 days of SRT, and variable HRT ranging from 20 to 6h. The effects of the influent COD/SO?-S ratio (ranging from 2 to 12) and the MLTS concentration (ranging from 6 to 22 g L?1) were also analysed. The main performance results were about 87% of COD removal, effluent VFA below 20 mg L?1 and biogas methane concentrations over 55% v/v. Methane yield was strongly affected by the influent COD/SO?-S ratio. No irreversible fouling problems were detected, even for MLTS concentrations above 22 g L?1.     

Retention of small charged impurities during ultrafiltration

Ultrafiltration is used to remove small impurities from a variety of processing streams. However, the clearance of small charged impurities may be inadequate due to electrostatic exclusion by the charged ultrafiltration membranes, an effect that has been largely unappreciated. Ultrafiltration experiments were performed to evaluate the transmission of several model impurities with different electrical charge through ultrafiltration membranes having different surface charge characteristics. Highly charged impurities are strongly rejected by charged cellulose and polyethersulfone membranes even though these solutes are much smaller than the membrane pore size. These effects could be eliminated by using high ionic strength solutions to shield the electrostatic interactions. The sieving data are in good agreement with model calculations based on the partitioning of charged spheres into charged cylindrical pores. Guidelines are developed for estimating conditions needed to obtain effective removal of small charged impurities through charged ultrafiltration membranes.     

Comparison between a moving bed membrane bioreactor and a conventional membrane bioreactor on organic carbon and nitrogen removal

A membrane bioreactor filled with carriers instead of activated sludge named a moving bed membrane bioreactor (MBMBR) was investigated for simultaneously removing organic carbon and nitrogen in wastewater. Its performance was compared with a conventional membrane bioreactor (CMBR) at various influent COD/TN ratios of 8.9–22.1. The operational parameters were optimized to increase the treatment efficiency. COD removal efficiency averaged at 95.6% and 96.2%, respectively, for MBMBR and CMBR during the 4 months experimental period. The MBMBR system demonstrated good performance on nitrogen removal at different COD/TN ratios. When COD/TN was 8.9 and the total nitrogen (TN) load was 7.58 mg/l h, the TN and ammonium nitrogen removal efficiencies of the MBMBR were maintained over 70.0% and 80.0%, respectively, and the removed total nitrogen (TN) load reached to 5.31 mg/l h. Multifunctional microbial reactions in the carrier, such as simultaneous nitrification and denitrification (SND), play important roles in nitrogen removal. In comparison, the CMBR did not perform so well. Its TN removal was not stable, and the removed total nitrogen (TN) load was only 1.02 mg/l h at COD/TN ratio 8.9. The specific oxygen utilization rate (SOUR) showed that the biofilm has a better microbial activity than an activated sludge. Nevertheless, the membrane fouling behavior was more severe in the MBMBR than in the CMBR due to a thick and dense cake layer formed on the membrane surface, which was speculated to be caused by the filamentous bacteria in the MBMBR.     

Purification of a conjugated polysaccharide vaccine using tangential flow diafiltration

Conjugated vaccines prepared from the capsular polysaccharide of Streptococcus pneumoniae can provide immunization against invasive pneumococcal disease, meningitis, and otitis media. One of the critical steps in the production of these vaccines is the removal of free (unreacted) polysaccharides from the protein-polysaccharide conjugate. Experimental studies were performed to evaluate the effects of membrane pore size, filtrate flux, and solution conditions on the transmission of both the conjugate and free polysaccharide through different ultrafiltration membranes. Conjugate purification was done using diafiltration performed in a linearly-scalable tangential flow filtration cassette. More than 98% of the free polysaccharide was removed within a 5-diavolume diafiltration process, which is a significant improvement over previously reported results for purification of similar conjugated vaccines. These results clearly demonstrate the opportunities for using ultrafiltration/diafiltration for the final purification of conjugated vaccine products.     

Tangential flow microfiltration and ultrafiltration for human influenza A virus concentration and purification

Large scale purification of viruses and viral vectors for gene therapy applications and viral vaccines is a major separation challenge. Here tangential flow microfiltration and ultrafiltration using flat sheet membranes has been investigated for concentration of human influenza A virus. Ultrafiltration membranes with molecular weight cutoffs of 100 and 300 kDa as well as 0.1, 0.2 and 0.45 microm microfiltration membranes have been tested. The results indicate that use of 300 kDa membranes not only concentrate the virus particles but also lead to a significant removal of host cell proteins and DNA in the permeate. Tangential flow filtration may be used to fractionate virus particles. Human influenza A virus particles are spherical with an average size of 100 nm. Use of a 0.1 microm membrane leads to passage of virus particles less than 100 nm into the permeate and an increase of larger particles in the retentate. These results suggest that control of the transmembrane pressure, membrane pore size and pore size distribution could enable isolation of intact virus particles from damaged virions. Isolation of the virus particles of interest from viral fragments and other particulate matter could result in simplification of subsequent purification steps. Larger pore size membranes such as 0.45 microm that allow the passage of all virus particles may be used to remove host cell fragments. In addition virus particles attached to these fragments will be removed. Careful selection of membrane morphology and operating conditions will be essential in order to maximize the benefit of tangential flow filtration steps in the purification of viral products from cell cultures.     

Determination of size distribution and encapsulation efficiency of liposome-encapsulated hemoglobin blood substitutes using asymmetric flow field-flow fractionation coupled with multi-angle static light scattering

In this study, we investigated the size distribution, encapsulation efficiency, and oxygen affinity of liposome-encapsulated tetrameric hemoglobin (LEHb) dispersions and correlated the data with the variation in extruder membrane pore size, ionic strength of the extrusion buffer, and hemoglobin (Hb) concentration. Asymmetric flow field-flow fractionation (AFFF) in series with multi-angle static light scattering (MASLS) was used to study the LEHb size distribution. We also introduced a novel method to measure the encapsulation efficiency using a differential interferometric refractive index (DIR) detector coupled to the AFFF-MASLS system. This technique was nondestructive toward the sample and easy to implement. LEHbs were prepared by extrusion using a lipid combination of dimyristoyl-phosphatidylcholine, cholesterol, and dimyristoyl-phosphatidylglycerol in a 10:9:1 molar ratio. Five initial Hb concentrations (50, 100, 150, 200, and 300 mg Hb per mL of buffer) extruded through five different membrane pore diameters (400, 200, 100, 80, and 50 nm) were studied. Phosphate buffered saline (PBS) and phosphate buffer (PB) both at pH 7.3 were used as extrusion buffers. Despite the variation, extrusion through 400-nm pore diameter membranes produced LEHbs smaller than the pore size, extrusion through 200-nm membranes produced LEHbs with diameters close to the pore diameter, and extrusion through 100-, 80-, and 50-nm membranes produced LEHbs larger than the pore sizes. We found that the choice of extrusion buffer had the greatest effect on the LEHb size distribution compared to either Hb concentration or extruder membrane pore size. Extrusion in PBS produced larger LEHbs and more monodisperse LEHb dispersions. However, LEHbs extruded in PB generally had higher Hb encapsulation efficiencies and lower methemoglobin (metHb) levels. The choice of extrusion buffer also affected how the encapsulation efficiency correlated with Hb concentration, extruder pore size, and the metHb level. The most optimum encapsulation efficiency and amount of Hb entrapped were achieved at the highest Hb concentration and the largest pore size for both extrusion buffers (62.38% and 187.14 mg Hb/mL of LEHb dispersion extruded in PBS, and 69.98% and 209.94 mg Hb/mL of LEHb dispersion extruded in PB). All LEHbs displayed good oxygen-carrying properties as indicated by their P(50) and cooperativity coefficients. LEHbs extruded in PB had an average P(50) of 23.04 mmHg and an average Hill number of 2.29, and those extruded in PBS had average values of 27.25 mmHg and 2.49. These oxygen-binding properties indicate that LEHbs possess strong potential as artificial blood substitutes. In addition, the metHb levels in PB-LEHb dispersions are significantly low even in the absence of antioxidants such as N-acetyl-L-cysteine.