A two-stage ultrafiltration and nanofiltration process for recycling dairy wastewater

A two-stage ultrafiltration and nanofiltration (UF/NF) process for the treatment of model dairy wastewater was investigated to recycle nutrients and water from the wastewater. Ultracel PLGC and NF270 membranes were found to be the most suitable for this purpose. In the first stage, protein and lipid were concentrated by the Ultracel PLGC UF membrane and could be used for algae cultivation to produce biodiesel and biofuel, and the permeate from UF was concentrated by the NF270 membrane in the second stage to obtain lactose in retentate and reusable water in permeate, while the NF retentate could be recycled for anaerobic digestion to produce biogas. With this approach, most of dairy wastewater could be recycled to produce reusable water and substrates for bioenergy production. Compared with the single NF process, this two-stage UF/NF process had a higher efficiency and less membrane fouling.     

Downstream separation and purification of bio-based alpha-ketoglutaric acid from post-fermentation broth using a multi-stage membrane process

In this study, a multi-stage membrane process, assisted by vacuum evaporation and crystallization, for recovery of bio-based alpha-ketoglutaric acid from the actual post-fermentation broth was designed and investigated. In the first part of this study, pre-treatment of crude fermentation broth (centrifugation-ultrafiltration-nanofiltration) was carried out to remove biomass, proteins, sugars, part of inorganic ions and color compounds. The commercial ceramic UF membrane (15 kDa) and nanofiltration ceramic membrane (200 Da or 450 Da) were applied. Electrodialysis with a bipolar membrane was proposed for separation of ionic compounds and simultaneous electro-acidification to the acid form. During bipolar membrane electrodialysis carried out under acidic conditions, it was possible to remove close to 90 % of alpha-ketoglutaric acid. Moreover, the migration of other acids present in the fermentation broth (lactic and acetic) was significantly limited. The calculated specific energy consumption was low and equal to 0.6 kW h/kg. The final purification using crystallization assisted vacuum evaporation allowed obtaining alpha-ketoglutaric acid in solid form. Analysis of the final product showed that the proposed method of alpha-ketoglutaric acid recovery gives the acid of high purity equal to 94.8 %. Furthermore, the presented results have practical relevance and may in future be the basis for the development of separation technologies of alpha-ketoglutaric acid from the fermentation broth on industrial scale.     

Purification of monoclonal antibodies derived from transgenic goat milk by ultrafiltration

With the goal of recovering heterologous immunoglobulin (IgG), which comprises 10-15% of the total proteins, from transgenic goat milk at 80% yield and 80% purity, we have developed and tested a two-step membrane isolation and purification process. In the first step, reported earlier by Baruah and Belfort, microfiltration was used to fractionate the milk proteins and recover > 90% of the original IgG at a purity of about 15-20% in the permeate stream. Here, we focus on ultrafiltration (UF) to increase the purity of the target protein to 80%, while maintaining a relatively high IgG yield (80%). Tangential flow UF experiments in diafiltration mode were conducted with 100 kDa cellulosic membranes to evaluate the optimal pH, ionic strength, and uniform transmembrane pressure (TMP). The TMP was kept uniform by permeate circulation in co-flow mode. The traditional approach of conducting the UF process close to the pI of the predominant whey proteins (15-40 kDa, pI 5.2), to transmit these proteins while retaining heterologous IgG (155 kDa), could not be applied here because of precipitation of residual casein at pH values lower than 8.5. Instead, the packing characteristics of the cake layer on the membrane wall, as elucidated in the Aggregate Transport Model presented by Baruah et al. was utilized to achieve a selectivity of > 15, which was sufficient to meet the stated goals of purity and yield for this difficult separation. This combined process is expected to reduce the load on subsequent purification and polishing steps for eventual therapeutic use.     

New method for exopolysaccharide determination in culture broth using stirred ultrafiltration cells

A new method to remove simple carbohydrates from culture broth prior to the quantification of exopolysaccharides (EPS) was developed and validated for the EPS-producing strain, Lactobacillus rhamnosus RW-9595M. This method uses ultrafiltration (UF) in stirred cells followed by polysaccharide detection in the retentate by the phenol-sulfuric acid method. The UF method was compared with a conventional method based on ethanol extraction, dialysis, protein removal by trichloroacetic acid (TCA) and freeze-drying. EPS production during pH-controlled batch fermentations in basal minimum medium, whey permeate (WP). and whey permeate supplemented with yeast extract, minerals and Tween-80 (SWP) was determined by the new UF and conventional methods. EPS recovery by the new method ranged from 83% to 104% for EPS added in the concentration range 40-1,500 mg/l in 0.1 M NaCl solution or culture medium. The UF method was rapid (8 h), accurate and simple, and required only a small sample volume (1-5 ml). A very high maximum EPS production was measured in SWP by both the UF and conventional methods (1,718 and 1,755 mg/l).     

Concentration and desalting by membrane processes of a natural pigment produced by the marine diatom Haslea ostrearia Simonsen

The marine microalga Haslea ostrearia, also called blue navicula, presents the unique peculiar property among the diatoms, to produce at its extremities a blue hydrosoluble pigment called marennine. It is presented the concentration and the desalting of the exocellular pigment by membrane processes (ultrafiltration, nanofiltration, reverse osmosis). Nanofiltration is particularly developed given the potential of this type of application both for the concentration of molecules and for desalting. It is shown the effect of velocity and pressure on performances of nanofiltration membranes. Permeation flux superior to 100 l h⁻¹ m⁻² (at 14.10⁵ Pa) are obtained with the Kiryat Weizmann membrane MP 20 (polyester coated with a polyacrylonitrile layer, cut-off 450 Da). For the desalting of the blue pigment solution, nanofiltration membranes present a few advantages: a low salt rejection (less than 10% at 14.10⁵ Pa) and a high pigment rejection (the nanofiltration membrane MP 20 retains more than 95% of the pigment). This membrane used in diafiltration mode allows an acceptable speed of desalting (700 g of salt eliminated per hour and per m² at 25.10⁵ Pa for a concentration of 18 g of salt per litre of solution).     

有机溶液环境中的迷迭香酸纳滤分离行为及富集工艺

本文采用纳滤分离低浓度乙醇水溶液中迷迭香酸,分析其分离行为并探索富集可行性。结果表明:乙醇浓度对纳滤膜通量和迷迭香酸截留率均有影响,通过响应面法建立二次回归模型,在保障分离效率及截留率的前提下,优化参数为溶质浓度0.41 mg/mL,乙醇浓度20.00%,截留分子量450 Da,迷迭香酸截留率93.81%,预测值较接近与理论值误差较小,数学模型准确可行。通过工艺对比,纳滤富集工艺相较于传统减压浓缩,迷迭香酸保留率提高35.1%,技术优势明显。结果还表明,乙醇水溶液环境下,长周期使用纳滤膜存在溶胀或污染的不可逆性,迷迭香酸截留率未发生明显变化,说明纳滤膜组件分离性能的耐用性较好。     

Concentration of 6-aminopenicillanic acid from penicillin bioconversion solution and its mother liquor by nanofiltration membrane

In this study, nanofiltration was applied to the concentration of the 6-aminopenicillinic acid (6-APA) from bioconverted penicillin solution and also to its mother liquor. The 6-APA in the solution was concentrated from 0.211 mol/L to 0.746 mol/L by nanofiltration. The final maximum concentration was 3.6 times higher than the initial concentration and the recovery yield was 97% to 99% of the original 6-APA. The concentrated solution was crystallized with the yields of 88.9–90.2% and the purity of the crystallized product was about 98%. The concentration of 6-APA in the mother liquor after crystallization was 0.014 mol/L and thus was concentrated 20–30 fold by nanofiltration and crystallization. The recovery of 6-APA was over 98%. The salts contained in the mother liquor, such as NH₄Cl and KCl, could be removed by allowing them to permeate through the membrane.     

Effect of extraction temperature on the diffusion coefficient of polysaccharides from Spirulina and the optimal separation method

The extraction temperature had a significant impact on the concentration of polysaccharides derived from solid-liquid extraction of Spirulina. The polysaccharide concentration was significantly higher when the extraction was performed at 90°C than when it was performed at 80, 70, and 50°C. This result is related to the diffusion coefficients of the polysaccharides, which increased from 1.07 × 10^?12 at 50°C to 3.02 × 10^?12 m²/sec at 90°C. Using the Arrhenius equation, the pre-exponential factor (D ₀ ) and the activation energy (E _a ) for Spirulina polysaccharide extraction were calculated as 7.958 × 10^?9 m²/sec and 24.0 kJ/mol, respectively. Among the methods used for the separation of Spirulina polysaccharides, cetyltrimethylammonium bromide (CTAB, method I) and organic solvent (ethanol, in methods II and III) provided similar yields of polysaccharides. However, the separation of polysaccharides using an ultrafiltration (UF) process (method III) and ethanol precipitation was superior to separation via CTAB or vacuum rotary evaporation (method II). The use of a membrane with a molecular weight cut-off (MWCO) of 30 kDa and an area of 0.01 m² at a feed pressure of 103 kPa with a mean permeate flux of 39.3 L/m²/h and a retention rate of 95% was optimal for the UF process. The addition of two volumes (v/v) of ethanol, which gave a total polysaccharide content of approximately 4% dry weight, was found to be most suitable for polysaccharide precipitation. The results of a Sepharose 6B column separation showed that the molecular weights of the polysaccharides in fractions I and II were 212 and 12.6 kDa, respectively.     

Sample preparation in separation of the extracellular chitinolytic enzymes of the human intestinal bacterium Clostridium paraputrificum J4 from the culture fluids

Membrane ultrafiltration (UF) was used in sample preparation of the culture fluids of the human intestinal bacterium Clostridium paraputrificum strain J4 containing seven extracellular chitinolytic isoenzymes (38-90 kDa). The subsequent filtration of the bacteria-free supernatants was carried out through Millipore membranes with cut-off 100 and 30 kDa for separation of undigested components of the culture medium and bacterial metabolites with molecular weight higher and lower than that of the target enzymes. The chitinolytic enzymes, which were the minor components in the culture fluids, were concentrated at UF as well. The aim of the research consisted in evaluation of the effect of component composition of bacteria-free supernatants and the chemical nature of membrane active layer on partial fractionation of the chitinolytic enzymes, their recovery in retentates and purification degree. On the basis of the obtained experimental results, the sample preparation procedure of the culture fluids of C. paraputrificum J4 was established to be used further in chromatographic separations of the chitinolytic enzymes.     

Electroelution of fixed and stained membrane proteins from preparative sodium dodecyl sulfate-polyacrylamide gels into a membrane trap

The membrane trap is a new device for the electroelution of all kinds of charged macromolecules from gels. Instead of dialysis membranes, the membrane trap uses a new membrane. Retention of macromolecules in an electric field by dialysis membranes depends on the presence of sodium dodecyl sulfate (SDS) in the buffer. The new membrane retains all charged macromolecules larger than approximately 5000 Da without adsorbing them, independent of the use of SDS. Here we report the electroelution of five different lipophilic membrane proteins (33 to 193 kDa) of Mycoplasma pneumoniae from preparative SDS-polyacrylamide gels into a 300-microliter recovery volume. After an 8-h elution period, recovery ranged from 80 (193 kDa) to 97% (33 kDa). The "losses" were generally due to proteins still remaining in the gel slice. All of the eluted proteins tested in a dot-blot assay proved to be antigenically active. The advantages of the device described here are easy handling (insertion of membranes, open system), quantitative recovery, and high reproducibility of the elution results.     

利用乳化液膜分离丙氨酸的研究

对乳化液膜技术分离丙氨酸进行了实验研究,从而探索了从味精生产过程的。第二次等电点结晶母液”中回收丙氨酸的新途径。采用二(2-乙基已基)磷酸为载体和表面活性剂失水山梨醇单油酸酯、磺化煤油组成液膜体系。研究了影响乳化液膜提取的各种因素,确定了适宜的分离条件。丙氨酸的单级提取率超过60％,浓缩3倍以上。对该液膜体系在交流高压静电场作用下,箱式破乳器的破乳过程,建立了破乳速率和相关操作因素间的定量关系。     

Ultrafiltration of fermented broths and solvent extraction of antibiotics

Experiments of ultrafiltration with diafiltration were performed on fermented broths of benzylpenicillin and clavulanic acid produced at pilot scale at CIPAN, SA to achieve solid/liquid separation, antibiotic recovery and soluble protein retention. This study was done using a laboratorial/pilot scale ultrafiltration facility from Paterson Candy International Ltd. whose modules have a tubular configuration. PVDF membranes with a molecular weight cut-off of 100 000 (FP10) and 200 000 (FP200) were assessed. Comparison between solvent extraction of benzylpenicillin and clavulanic acid from filtered broth and ultrafiltered broth was also done. It was concluded that ultrafiltration with diafiltration of fermented broths of antibiotics using the FP10 membrane is a valid alternative to the use of rotary vacuum filters, flocculants and filter aid, deserving a further study to establish the best operating conditions. An economic appraisal also seems to be of paramount importance.To CIPAN-SA in which this work was done and where the fermented broths were produced. To JNICT/FSE for the funding of the project in the scope of which this work was done.     

Effects of culture density,conditioned medium and feeder cultures on microspore embryogenesis in Brassica napus L. cv. Topas

Summary In microspore cultures of Brassica napus L. cv. Topas, embryo yield increases with culture density up to about 40,000 microspores per ml. A much higher density (100,000 per ml) appears inhibitory to embryogenesis. A relatively high culture density (30,000 or 40,000 per ml) for the first 2–4 days of culture is crucial for embryogenesis, after which cultures may be diluted to allow better embryo growth.Medium conditioned by culturing microspores at 30,000 or 40,000 per ml for 1 day improved microspore-embryo yield in low density cultures (3,000 or 4,000 per ml) more than 3-fold. In contrast, media conditioned with microspores from 1–4 days or 0–4 days of culture were inhibitory.Use of feeder cultures resulted in up to 10-fold increase of embryo yield in low density microspore cultures, depending on the method used. Filter papers and other membranes placed on top of feeders greatly inhibited embryogenesis in the feeder layer as well as microspores cultured on the feeder, possibly due to poorer gaseous exchange.     

Effects of colloidal fouling and gas sparging on microfiltration of yeast suspension

Cross-flow microfiltration is an important step in separating Baker’s yeast (Saccharomyces cerevisiae) from aqueous suspension in many processes. However the permeate flux often declines rapidly due to colloidal fouling of membranes and concentration polarisation. The present work explores the possibility of maintaining acceptable permeate flux by co-current sparging of gas along with the feed, which would scour away colloidal deposits and reduce concentration polarisation of membranes. In this work, both washed and unwashed yeast were used to study the effect of washing to reduce protein fouling of membranes. It was found that permeate flux increased by 45% for liquid throughput of 75 kg/h for a feed concentration of 2.0 kg/m³ of washed yeast as compared with unwashed yeast suspension without gas sparging. For washed yeast suspension, the increase in gas flow rate from 0.5 lpm to 1.5 lpm (30 l/h to 90 l/h) had beneficial effect on permeate flux. It is concluded that in the present case, the gas flow rate should be less than or equal to the liquid flow rate for enhancement of permeates flux.     

Routes of nonelectrolyte permeability in gallbladder. Effects of 2,4,6- triaminopyrimidinium (TAP)

The organic cation 2,4,6-triaminopyrimidinium (TAP), which blocks the tight junction channels for cation permeability across gallbladder, also inhibits gallbladder permeability (P) to urea and glycerol without significantly affecting P to Cl(-), sucrose, 1,7-heptanediol, or water (osmotic or diffusional permeabilities). These effects together with the comparisons of P's in frog with P's in rabbit gallbladder suggest that sucrose migrates exclusively through the leakage pathway (through where Cl(-) permeates), and that urea and glycerol permeate in addition through both, the tight junction channels for cations and a polar pathway at the cell membranes. Water and 1,7-heptanediol probably permeate mainly through the epithelial cell membranes.     

Sugar Syrup Concentration Using Reverse Osmosis Membranes

In sugar manufacturing industries, initially dilute syrup is obtained from the cane sugar or beetroot, which should be concentrated. In many factories, sugar syrup concentration is carried out using evaporation. This process has two main problems. Firstly, it consumes a huge amount of energy due to high latent heat of water and secondly, heating may decompose the sugar molecules resulting in low‐quality and dark‐colored sugar. Low energy consuming reverse osmosis may be employed for concentrating sugar syrup without decomposing the molecules, resulting in high‐quality sugar with low cost. In this study different commercial reverse osmosis membranes (DS, DSII, PVD, FT30, BW30) and one nanofiltration membrane (NF45) were used for sugar syrup concentration. The results show that nanofiltration NF45 membrane has no effect on sugar syrup concentration. The rejections of sugar using DSII and PVD reverse osmosis membranes vary between 23 % and 33 % for different operating conditions. DS membrane rejected around 10 % of the sugar molecules in best conditions. FT30 membrane initially showed better performance (55 %). However, the rejection was decreased during time (minimum 7 %). For BW30 membrane, the rejection of sugar was better (60 %) compared to the other membranes used in this work. For two‐stage processes (i.e. the permeate of the first stage used as a feed for the second stage) the highest rejection (88 %) was obtained.     

The effects of alpha-lactabumin and whey protein concentrate on dry matter recovery, TCA soluble protein levels, and peptide distribution in the rat gastrointestinal tract

The effects of two dietary proteins on dry matter recovery, trichloroacetic acid (TCA) soluble protein concentration, and peptide distribution in gastrointestinal contents were investigated in rats trained to consume, in a single 2-hour daily meal, diets containing alpha-lactalbumin (alpha-LA) or whey protein concentrate (WPC) for two weeks. Compared with the WPC diet, the alpha-LA diet emptied faster from the stomach. Dry matter recovery was higher in the stomach contents of rats fed the WPC diet than in those given the alpha-LA diet, but dry matter content in the small intestine was comparable. TCA soluble protein levels in the stomach and the small intestinal contents were also significantly (P < 0.001) higher in rats fed the WPC diet. The concentration of peptides having molecular weights (MW) ranging from 12,500-30,000 daltons (Da) was higher in the stomach contents of rats fed the WPC diet. Conversely, the level of peptides ranging from 5000-12,500 Da was higher in the stomach contents of rats fed the alpha-LA diet. For both diets, the small intestinal contents were characterized by high levels of amino acids and small peptides. These results suggest that the hydrolysis and absorption of alpha-LA is faster than that of WPC.     

Fully integrated L-phenylalanine separation and concentration using reactive-extraction with liquid-liquid centrifuges in a fed-batch process with<Emphasis Type="Italic"> E. coli</Emphasis>

A novel in situ product recovery (ISPR) approach for the (fully) integrated separation of L-phenylalanine (L-phe) from a 20 l fed-batch process with the recombinant L-tyrosine auxotrophic strain E. coli F-4/pF81 is presented. The strain was rationally constructed for the production of the aromatic amino acid. Glucose and tyrosine control is used. A reactive extraction system consisting of kerosene, the cation-selective carrier D₂EHPA and sulphuric acid, all circulating in liquid-liquid centrifuges, is applied for the on-line L-phe separation from cell- and protein-free permeate. Permeate is drained off from the bioreactor bypass. Using the novel ISPR approach, a significantly extended product formation period at 0.25 mmol/(g*h) together with a reduced by-product formation and a 28% relative glucose/L-phe yield increase is observed. Thus, the ISPR approach is superior to the reference non-ISPR process and even offers extraction rates approximately three times higher than the published membrane-based process.     

Use of stable emulsion to improve stability, activity, and enantioselectivity of lipase immobilized in a membrane reactor

The enantiocatalytic performance of immobilized lipase in an emulsion membrane reactor using stable emulsion prepared by membrane emulsification technology was studied. The production of optical pure (S)-naproxen from racemic naproxen methyl ester was used as a model reaction system. The O/W emulsion, containing the substrate in the organic phase, was fed to the enzyme membrane reactor from shell-to-lumen. The enzyme was immobilized in the sponge layer (shell side) of capillary polyamide membrane with 50 kDa cut-off. The aqueous phase was able to permeate through the membrane while the microemulsion was retained by the thin selective layer. Therefore, the substrate was kept in the enzyme-loaded membrane while the water-soluble product was continuously removed from the reaction site. The results show that lipase maintained stable activity during the entire operation time (more than 250 h), showing an enantiomeric excess (96 +/- 2%) comparable to the free enzyme (98 +/- 1%) and much higher compared to similar lipase-loaded membrane reactors used in two-separate phase systems (90%). The results demonstrate that immobilized enzymes can achieve high stability as well as high catalytic activity and enantioselectivity.     

Separation of monoclonal antibody alemtuzumab monomer and dimers using ultrafiltration

This article examines the feasibility of using ultrafiltration to separate the monomer of the monoclonal antibody alemtuzumab (Campath or Campath-1H) from a mixture of dimer and higher-order oligomers (collectively called "dimers" here). Using parameter scanning ultrafiltration, we initially assessed the suitability of the following membranes: 100 kDa and 300 kDa polyethersulfone (PES) membranes, and a 100 kDa polyvinylidene fluoride (PVDF) membrane. A detailed study was then carried out to examine the effects of operating conditions (such as solution pH, ionic strength, stirring speed, and permeate flux) on the separation of the monomer from the dimers using 300 kDa PES and 100 kDa PVDF membranes. Results of the experiments carried out in the carrier phase ultrafiltration (CPUF) mode indicate that the size-based protein-protein separation critically depends on the membrane used as well as the system hydrodynamics. The separation of the monoclonal antibody monomer and dimers using 100 kDa PVDF membranes in the diafiltration mode was also examined. Experimental results demonstrate that under suitable conditions, it is feasible to obtain the alemtuzumab monomer with a purity of more than 93% and a yield of more than 85% (from a mixture of 75% monomer and 25% dimers, which is the typical composition obtained after affinity chromatography). Simulation study indicates that this could be further improved to a purity of more than 96% and a monomer yield of more than 96% by increasing the selectivity of separation or by employing a two-stage diafiltration process.