Removal of emerging trace organic contaminants by MBR-based hybrid treatment processes

The aim of this study was to demonstrate the complementarity of combining membrane bioreactor (MBR) treatment with UV oxidation or high pressure membrane filtration processes such as nanofiltration (NF) or reverse osmosis (RO) for the removal of trace organic contaminants (TrOC). The results suggest that the removal mechanisms of TrOC by either UV oxidation or NF/RO membrane filtration differ significantly from those of an MBR system. Thus, they can complement MBR treatment very well to significantly improve the removal of TrOC. MBR treatment can effectively remove hydrophobic and readily biodegradable hydrophilic TrOC. The remaining hydrophilic and biologically persistent TrOC were shown to be effectively removed by either UV oxidation or NF/RO membrane filtration. The combination of MBR with UV oxidation or NF/RO membrane filtration resulted in a removal ranging from 85% to complete removal (or removal to below the analytical detection limit) of all 22 TrOC selected in this study. In particular, it is noteworthy that although MBR treatment and direct UV oxidation separately achieved low removal of carbamazepine (a widely reported problematic compound), the combination of these two processes resulted in more than 96% removal.     

Affinity precipitation of proteins by polyligands

A novel technique for affinity precipitation has been developed in which multimeric target proteins are precipitated as a result of network formation by polymer-conjugated ligands (polyligands). A polyligand precipitant for avidin was synthesized by conjugation of biotin to a polyacrylamide-based backbone. The effects of mixing conditions, ligand substitution frequency, and molecular weight on affinity precipitation were examined using the biotin-PAAm precipitant. Biotin was replaced by iminobiotin to study the effect of the ligand-protein dissociation constant o affinity precipitation. The iminobiotin-PAAm precipitant was also used to examine the reversibility of the precipitation and recovery of the target protein after precipitation. (c) 1993 Wiley & Sons, Inc.     

Affinity precipitation of monoclonal antibodies by nonstoichiometric polyelectrolyte complexes

The nonstoichiometric polyelectrolyte complex (PEC) formed by poly(methacrylic acid) (degree of polymerization 1830) (PMAA)and poly(N-ethyl-4-vinyl-pyridinium bromide) (degree of polymerization 530) (PEVP) undergoes reversible precipitation from aqueous solution at any desired pH-value in the range 4.5–6.5 depending on the ionic strength and PEVP/PMAA ratio in the complex. The antigen, inactivated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit was covalently coupled to PEVP. The resulting GAPDH–PEVP/PMAA complex was used for the purification of antibodies from a 6G7 clone specific towards inactivated GAPDH. The crude extract was incubated with GAPDH-containing PEC and the precipitation of the PEC was carried out at 0.01 M NaCl and pH 4.5, 5.3, 6.0 and 6.5 using PEC with PEVP/PMAA ratios of 0.45, 0.3, 0.2 and 0.15, respectively. Purified antibodies were eluted at pH 4.0 where PECs of all compositions used were insoluble.PEC precipitation is accompanied only by small nonspecific coprecipitation of proteins. Precipitated PEC could be dissolved at pH 7.3 and used repeatedly. This revised version was published online in July 2006 with corrections to the Cover Date.     

Affinity precipitation of an antibody by ligand-modified phospholipids

A new method for the selective precipitation of proteins is applied to the isolation and purification of an antibody. Ligand-modified phospholipids (LMPs) are solubilized by the nonionic ethoxylated alcohol detergent, resulting in small (50 to 100 A) micellar aggregates of LMPs and surfactant. When introduced into protein solutions containing an antibody for which the LMP has specific affinity, the ligand binds to the protein. Hydrophobic interactions between phospholipid tail groups bound to the protein molecules result in an insoluble precipitate. Polyclonal and monoclonal antibiotin antibody (pABA and mABA) are shown to be selectively precipitated using ratios of dimyristoylphosphatidylethanolamidobiotin (DMPE-B) to ABA ranging from 1:1 to 19:1. The kinetics and yield of the precipitation achieve a maximum at a ratio of DMPE-B to ABA of approximately 7:1. The kinetics and magnitude of the turbidity change are modeled using the Mie theory of light scattering coupled with the smoluchowski theory of aggregation. The kinetics are shown to be enhanced significantly by the addition of salt. In particular, the addition of 0.5 M ammonium sulfate salt increases the rate of precipitation by more than an order of magnitude. It is demonstrated that pABA can be recovered with total activity yields of 60% to 70% from mixtures containing nonspecific lgG antibodies in very high purity (>99%). (c) 1994 John Wiley & Sons, Inc.     

A protein network for phospholipid synthesis uncovered by a variant of the tandem affinity purification method in Escherichia coli

In prokaryotes, acyl carrier protein (ACP) is a cofactor central to a myriad of syntheses, including fatty acid and phospholipid synthesis. To fulfill its function, ACP must therefore interact with a multitude of different enzymes, which includes the thioesterase YbgC. We found a specific interaction between ACP and YbgC whose thioesterase activity has been demonstrated in vitro on acyl-CoA derivatives, but whose physiological function in bacteria remains unknown. Therefore, YbgC could be a thioesterase active on some specific acyl-ACPs. We then assigned a function to the ACP/YbgC pair by employing a proteomic approach derived from tandem affinity purification, the split tag method. This technique allowed us to purify proteins interacting with ACP and YbgC proteins at the same time. Interactions with PlsB, a sn-glycerol-3-phosphate acyltransferase and PssA, a phosphatidylserine synthase, were identified and validated, showing that YbgC is involved in phospholipid metabolism. Furthermore, using an in vivo bacterial two-hybrid interaction analysis, we showed for the first time that enzymes of the phospholipid synthesis pathway form a complex in the inner membrane. Taken together, these results describe an integrated protein network that could be involved in the coordination of phospholipid metabolism.     

A 13C-NMR study of 10,12-tricosadiynoic acid and the corresponding phospholipid and phospholipid polymer

Diacetylene phospholipids are presently being studied because of their potential to polymerise in vesicles, multilayers and natural biomembranes. ¹³C-NMR spectra and spin-lattice relaxation times have now been obtained of a diacetylene phospholipid present in a sonicated dispersion in water. Similar data have been obtained of a monoacetylene phospholipid and a saturated phospholipid. For further comparison the spectrum of a diacetylenic fatty acid in $\text{benzene}\text{-d}{}_6$ was also examined and relaxation data obtained. A comparison of the various relaxation data provides an indication of the restricted motion associated with the two conjugated triple bonds of the diacetylene phospholipid within the lipid bilayer structure. A proximity interaction between diacetylene groups occurs and a conformation for the diacetylene part of the lipid in the bilayer is deduced. The ¹³C-NMR spectrum of a soluble phospholipid polymer in C²HCl₃, obtained by ultraviolet irradiation of the diacetylene phospholipid, shows that the two conjugated triple bonds of the monomer is replaced in the polymer by an alternating double and triple bonded conjugated structure.     

Metal chelate affinity precipitation of RNA and purification of plasmid DNA

The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine `tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.     

Selective antibody precipitation using polyelectrolytes: A novel approach to the purification of monoclonal antibodies

We evaluated the potential for polyelectrolyte induced precipitation of antibodies to replace traditional chromatography purification. We investigated the impact of solution pH, solution ionic strength and polyelectrolyte molecular weight on the degree of precipitation using the anionic polyelectrolytes polyvinylsulfonic acid (PVS), polyacrylic acid (PAA), and polystyrenesulfonic acid (PSS). As we approached the pI of the antibody, charge neutralization of the antibody reduced the antibody–polyelectrolyte interaction, reducing antibody precipitation. At a given pH, increasing solution ionic strength prevented the ionic interaction between the polyelectrolyte and the antibody, reducing antibody precipitation. With increasing pH of precipitation, there was an increase in impurity clearance. Increasing polyelectrolyte molecular weight allowed the precipitation to be performed under conditions of higher ionic strength. PVS was selected as the preferred polyelectrolyte based on step yield following resolubilization, purification performance, as well as the nature of the precipitate. We evaluated PVS precipitation as a replacement for the initial capture step, as well as an intermediate polishing step in the purification of a humanized monoclonal antibody. PVS precipitation separated the antibody from host cell impurities such as host cell proteins (HCP) and DNA, process impurities such as leached protein A, insulin and gentamicin, as well as antibody fragments and aggregates. PVS was subsequently removed from antibody pools to <1 µg/mg using anion exchange chromatography. PVS precipitation did not impact the biological activity of the resolubilized antibody. Biotechnol. Bioeng. 2009;102: 1141–1151. © 2008 Wiley Periodicals, Inc.     

Continuous capture of recombinant antibodies by ZnCl2 precipitation without polyethylene glycol

The capture of recombinant antibodies from cell culture broth is the first critical step of downstream processing. We were able to develop a precipitation‐based method for the capture and purification of monoclonal antibodies based on divalent cations, namely ZnCl₂. Traditional precipitation processes have to deal with high dilution factors especially for resolubilization and higher viscosity due to the use of PEG as precipitation or co‐precipitation agent. By the use of the crosslinking nature of divalent cations without the use of PEG, we kept viscosity from the supernatant and resolubilization dilution factors very low. This is especially beneficial for the solid–liquid separation for the harvest and wash of the precipitate in continuous mode. For this harvest and wash, we used tangential flow filtration that benefits a lot from low viscosity solutions, which minimizes the membrane fouling. With this precipitation based on ZnCl_2, we were able to implement a very lean and efficient process. We demonstrated precipitation studies with three different antibodies, Adalimumab, Trastuzumab, and Denosumab, and a continuous capture case study using tangential flow filtration for precipitate recovery. In this study, we achieved yields of 70%.     

Centrifugal precipitation chromatography

Centrifugal precipitation chromatography separates analytes according their solubility in ammonium sulfate (AS) solution and other precipitants. The separation column is made from a pair of long spiral channels partitioned with a semipermeable membrane. In a typical separation, concentrated ammonium sulfate is eluted through one channel while water is eluted through the other channel in the opposite direction. This countercurrent process forms an exponential AS concentration gradient through the water channel. Consequently, protein samples injected into the water channel is subjected to a steadily increasing AS concentration and at the critical AS concentration they are precipitated and deposited in the channel bed by the centrifugal force. Then the chromatographic separation is started by gradually reducing the AS concentration in the AS channel which lowers the AS gradient concentration in the water channel. This results in dissolution of deposited proteins which are again precipitated at an advanced critical point as they move through the channel. Consequently, proteins repeat precipitation and dissolution through a long channel and finally eluted out from the column in the order of their solubility in the AS solution. The present method has been successfully applied to a number of analytes including human serum proteins, recombinant ketosteroid isomerase, carotenoid cleavage enzymes, plasmid DNA, polysaccharide, polymerized pigments, PEG-protein conjugates, etc. The method is capable to single out the target species of proteins by affinity ligand or immunoaffinity separation.     

Purification of anti-bromelain antibodies by affinity precipitation using pNIPAm-linked bromelain

Affinity precipitation has emerged as a very useful technique for the purification of proteins. Here it has been employed for the purification of anti-bromelain antibodies from rabbit serum. A system has been developed for reversibly binding and thermoprecipitating antibodies. Anti-bromelain antibodies were raised in rabbit by immunizing it with bromelain. Poly-N-isopropylacrylamide (pNIPAm)–bromelain conjugate was prepared and incubated with rabbit serum. After that the temperature was raised for thermal precipitation of the polymer. Antibodies were then eluted from the complex by incubating it with a small volume of buffer, pH 3.0. This method is very effective in concentrating the antibodies. Purity and specificity of the antibodies were checked by gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. The study of the effect of pH and temperature on the binding of the antibodies to the conjugate showed that the optimum binding occurred at pH 8.0 and 25°C.The polymer enzyme conjugate was further used for another cycle.     

Surface activity and interaction of StarD7 with phospholipid monolayers

StarD7 protein forms stable Gibbs and Langmuir monolayers at the air-buffer interface showing marked surface activity. The latter is enhanced by penetration into phospholipid films at an initial surface pressure above the protein’s own equilibrium adsorption surface pressure to a lipid-free interface. The protein-phospholipid stabilizing interactions at the interface depend on the lipid, with preference for phosphatidylserine, cholesterol, and phosphatidylglycerol, and the increases of lateral surface pressure generated are comparable to those of other membrane-active proteins. The surface activity of StarD7 is strong enough to thermodynamically drive and retain StarD7 at the lipid membrane interface where it may undergo lipid-dependent reorganization as indicated by changes of surface pressure and electrostatics.     

Proinflammatory responses by glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum are mainly mediated through the recognition of TLR2/TLR1

The glycosylphosphatidylinositols (GPIs) of Plasmodium falciparum have been shown to activate macrophages and produce inflammatory responses. The activation of macrophages by malarial GPIs involves engagement of Toll like receptor 2 (TLR2) resulting in the intracellular signaling and production of cytokines. In the present study, we investigated the requirement of TLR1 and TLR6 for the TLR2 mediated cell signaling and proinflammatory cytokine production by macrophages. The data demonstrate that malarial GPIs, which contain three fatty acid substituents, preferentially engage TLR2–TLR1 dimeric pair than TLR2–TLR6, whereas their derivatives, sn-2 lyso GPIs, that contain two fatty acid substituents recognize TLR2–TLR6 with slightly higher selectivity as compared to TLR2–TLR1 heteromeric pair. These results are analogous to the recognition of triacylated bacterial and diacylated mycoplasmal lipoproteins, respectively, by TLR2–TLR1 and TLR2–TLR6 dimers, suggesting that the lipid portions of the microbial GPI ligands play essential role in determining their TLR recognition specificity.     

Removal of nutrients from piggery wastewater using struvite precipitation and pyrogenation technology

In this paper, removal of nutrients from piggery wastewater by struvite crystallization was conducted using a combined technology of low-cost magnesium source in struvite precipitation and recycling of the struvite pyrolysate in the process. In the present research, it was found that high concentrations of K⁺ and Ca²⁺ present in the solution significantly affected the removal of nutrients. When the struvite crystallization formed at the condition of dosing the magnesite pyrolysate at a Mg:N:P molar ratio of 2.5:1:1, and having a reaction time of 6 h, a majority of nutrients in piggery wastewater can be removed. Surface characterization analysis demonstrated that the main components of the pyrolysate of the obtained struvite were amorphous magnesium sodium phosphate (MgNaPO₄) and MgO. When the struvite pyrolysate was recycled in the process at the pH range of 8.0-8.5, the precipitation effect was optimum. When the struvite pyrolysate was recycled repeatedly at pH 8.5 or without any adjustment of pH, the outcome of the removal of the nutrients in both cases was similar. With the increase in the number of recycle times, the performance of struvite precipitation progressively decreased. An economic evaluation showed that the combination of using low-cost material and recycling of struvite was feasible. Recycling struvite for three process cycles could save the chemical costs by 81% compared to the use of pure chemicals.     

Large-scale purification of oligonucleotides by extraction and precipitation with butanole

Today the synthesis of oligonucleotides is a well-established process. Using automatic synthesizers even kilogram quantities can be produced in a few hours. However, the purification of the final product is still time-consuming and needs a complex apparatus. In this article, a simple and fast purification method for the large-scale syntheses of oligonucleotides is described. According to the method of Sawadago and van Dyke ([1991] Nucleic Acids Res 19:674-675) for small-scale oligonucleotide purification, oligonucleotides in mumol to mmol amounts were purified by liquid-liquid extraction using butanole as the extraction liquid. Choosing appropriate ratios of extraction liquid to oligonucleotide solution, simultaneous purification and precipitation could be achieved. It was found that the yield of the purified oligonucleotide was mainly affected by the temperature. Yield decreased with increasing temperature. The use of this improved extraction procedure allows the purification of gram to kilogram quantities of oligonucleotides in less than a day with simple equipment and high yield.     

有机污染环境的植物修复研究进展

分析了近年来国内外的文献资料,对有机污染物污染环境的植物修复研究进展作了综述。植物能通过根系从环境中吸收和积累PCBs、PAHs等有机污染物,并将吸收的TNT、TCE及有机农药降解为高极性产物、水和CO2;另一方面植物根际可促进有机污染物的根际生物吸收与,使植物对有机污染环境的修复效果更明显。文中探讨了有机污染环境的植物修复技术的优势、问题与未来的研究。     

Fractionation of protein, RNA, and plasmid DNA in centrifugal precipitation chromatography using cationic surfactant CTAB containing inorganic salts NaCl and NH(4)Cl

Centrifugal precipitation chromatography (CPC) is a separation system that mainly employs a moving concentration gradient of precipitating agent along a channel and solutes of interest undergo repetitive precipitation-dissolution, fractionate at different locations, and elute out from the channel according to their solubility in the precipitating agent solution. We report here for the first time the use of a CPC system for fractionation of protein, RNA, and plasmid DNA in clarified lysate produced from bacterial culture. The cationic surfactant cetyltrimethylammonium bromide (CTAB) was initially used as a precipitating agent; however, all biomolecules showed no differential solubility in the moving concentration gradient of this surfactant and, as a result, no separation of protein, RNA, and plasmid DNA occurred. To overcome this problem, inorganic salts such as NaCl and NH(4)Cl were introduced into solution of CTAB. The protein and RNA were found to have higher solubility with the addition of these salts and separated from the plasmid DNA. Decreasing surface charge density of CTAB upon addition of NaCl and NH(4)Cl was believed to lead to lower surfactant complexation, and therefore caused differential solubility and fractionation of these biomolecules. Addition of CaCl(2) did not improve solubility and separation of RNA from plasmid DNA.     

The binding of organic ions to phospholipid bilayers

The binding of organic anions and cations, mainly tetraphenylboride and tetraphenylarsonium, to phospholipid membranes has been studied using an NMR method. Binding is appreciable and is affected by cholesterol in the membrane and counterions in solution. The passage of the organic anions through the membrane has also been followed. These measurements indicate that it is naive to use organic anions to measure membrane potentials in a simple manner.     

Some microbial contaminants and control agents in a diet and larvae of Heliothis spp

Sixteen tests were conducted with four antimicrobial agents that are routinely incorporated into lepidopterous larval diets for bacterial and fungal inhibition. The agents tested in all possible combinations were benomyl, chlortetracycline, sorbic acid, and methyl P-hydroxybenzoate. Those combinations that contained benomyl and sorbic acid in the formula were effective in complete microbial suppression during the larval portion of the 35-day testing period. Insect development in plastic cups of test diets with benomyl and sorbic acid averaged 97 and 93% pupation and adult emergence, respectively. The 12 tests that did not contain benomyl and sorbic acid were contaminated with some of the following fungi: Aspergillus niger, A. flavus, Rhizopus nigricans, Cladosporium, Fusarium, yeasts, or the bacterium, Bacillus subtilis. Microorganisms in Heliothis spp. larvae from mass-rearing diets were tested, examined, and identified. Bacteria recovered from laboratory-reared Heliothis larvae included Pseudomonas aeruginosa, P. maltophilia, Micrococcus luteus, α-hemolytic Streptococcus, Serratia marcescens, and S. rubidaea. Tests were conducted with sensi-discs for the selection of antimicrobials to arrest contamination in the larval digestive systems.     

Polymerized liposome as ligand carrier for affinity precipitation of proteins

A polymerized liposome (PLS) was prepared using a synthesized phospholipid with a diacetylene moiety in the hydrophobic chain and an amino group in the hydrophilic head. The PLS was used as a novel ligand carrier for affinity precipitation of proteins because it showed a reversibly precipitable property on salt addition and removal. Soybean trypsin inhibitor (STI) was easily immobilized on the PLS by a one-step carbodiimide reaction. The PLS showed no nonspecific adsoprtion of proteins. It had a large ligand coupling capacity, and then a large adsorption capacity for trypsin after STI immobilization. The PLS with immpbilized STI was recycled three times for the purification of trypsin from a crude pancreatic extract. Although the degree of purification was compromised by the impurity of the STI employed, in each run the purification factor reached about 6 and more than 80% of trypsin activity was recovered. The results indicated that the PLS was a potential ligand carrier for affinity precipitation of proteins. (c) 1995 John Wiley & Sons, Inc.