Nickel accumulation by immobilized biofilm of Cirobacter sp. containing cell-bound polycrystalline hydrogen uranyl phosphate

Hydrogen uranyl phosphate (HUO2PO4: HUP), deposited enzymatically on Citrobacter N14 cells immobilized as biofilm on ceramic Raschig rings in a flow-through column, removed nickel quantitatively from dilute aqueous solution in the form of nickel uranyl phosphate, via intercalative ion exchange. Nickel-loaded columns were regenerated by washing either with citrate buffer or with buffer containing UO22 and phosphate donor (glycerol 2-phosphate), this giving additional crystalline HUP deposit for subsequent improvement of nickel removal. No uranium release occurred during selective desorption of Ni, proving the integrity of the biofilm within the column. The use of ceramic supports to manufacture an artificial, bioinorganic, ion exchanger is novel and the use of solid matrices overcomes the problems of mechanical stability which limit the applications of gel-immobilized cells for large-scale processes. © Rapid Science Ltd. 1998     

Microbially enhanced chemisorption of nickel into biologically synthesized hydrogen uranyl phosphate: a novel system for the removal and recovery of metals from aqueous solutions

Ni(2+) was removed quantitatively from aqueous flows by columns loaded with polycrystalline hydrogen uranyl phosphate (HUP) bound to immobilized cells of Citrobacter sp. The columns functioned effectively in Ni uptake/regeneration cycles; five cycles were completed without significant decrease in the Ni-removing capacity of the column. The influence of pH, temperature, and flow rate on the Ni-removing capacity of the columns was examined. The composition of the Ni/HUP cell-bound deposits was confirmed by X-ray diffraction analysis (XRD) and proton-induced X-ray emission (PIXE) spectroscopy following several consecutive metal challenges and is discussed in relation to the mechanism of Ni(2+) removal from solution via ion-exchange intercalation into the interlayer space of HUP. Ni was selectively recovered from the columns using citrate or tartrate. The regenerated columns functioned effectively in Ni removal throughout repeated Ni challenge and desorption cycles. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 319-328, 1997.     

Axonal surface charges: evidence for phosphate structure.

The effect of different extracellular alkaline-earth cations (Ca2+, Mg2+, Sr2+, Ba2+) upon the threshold membrane potential for spike initiation in crayfish axon has been studied by means of intracellular microelectrodes. This was done at the following extracellular concentrations of the divalent uranyl ion (UO2/2+): 1.0 X 10(-6) M, 3.0 X 10(-6) M, and 9.0 X 10(-6) M. At each concentration employed, extensive neutralization of axonal surface charges by UO2/2+ was evidenced by the fact that equal concentrations (50 mM) of alkaline-earth cations did not have the same effect on the threshold potential. The selectivity sequences observed at the different uranyl-ion concentrations were: 1.0 X 10(-6) M UO2/2+, Ca2+ greater than Mg2+ greater than Sr2+ greater than Ba2+; 3.0 X 10(-6) M UO2/2+, Ca2+ greater than Mg2+ greater than Ba2+ larger than or equal to Sr2+; 9.0 X 10(-6) M UO2/2+, Ca2+ approximately Ba2+ greater than Sr2+ greater than Mg2+. These selectivity sequences are in accord with the equilibrium selectivity theory for alkaline-earth cations. At each of the concentrations used, uranyl ion did not have any detectable effect on the actual shape of the action potential itself. It is concluded that many (if not most) of the surface acidic groups in the region of the sodium gates represent phosphate groups of membrane phospholipids, but that the m gates themselves are probably protein-aceous in structure.     

Structural characteristics of the saxitoxin receptor on nerve.

The effects of uranyl ion (UO22+; at low concentrations binds specifically to phosphate groups) and the cationic dye methylene blue (MB+; binds strongly to carboxyl groups) on saxitoxin (STX) potency in crayfish axon has been studied by means of intracellular microelectrodes. At pH 6.00 +/- 0.05 and 13.5 mM Ca2+, addition of 10.0 muM UO22+ + 5.0 nM STX had only slightly, if any, less effect on the spike's maximum rate of rise [0.79 +/- 0.04 (viz., mean +/- SEM) of control value] than did addition of 5.0 nM STX alone (0.72 +/- 0.05). Under the same conditions of pH and Ca2+ concentration, 1.0 mM MB+ had approximately the same effect: 1.0 mM MB+ + 5.0 nM STX, 0.76 +/- 0.03; 5.0 nM STX alone, 0.70 +/- 0.04. However, at pH 7.00 +/- 0.05 and lower Ca2+ concentrations, 1.0 mM MB+ significantly reduced STX potency. Using 6.0 mM Ca2+: 1.0 mM MB+ + 5.0 nM STX, 0.92 +/- 0.01; 5.0 nM STX alone, 0.68 +/- 0.08. Using 3.0 mM Ca2+, the corresponding values were 0.94 +/- 0.03 and 0.67 +/- 0.04. It is concluded that: (1) In accord with previous suggestions, the ionized acidic group known to exist in the Na channel (and to which a guanidinium group of STX appears to bind) is very likely a carboxyl group and not a phosphate group. (2) The accessible part of the Na channel mouth serving as the saxitoxin receptor probably does not include phospholipid in its structure proper.     

Ion exchange/complexation of the uranyl ion by Rhizopus biosorbent

Nonliving biomass of nine Rhizopus species effectively sequestered the uranyl ion from solution, taking up 150-250 mg U/g dry cells at 300 ppm U equilibrium concentration in solution, and 100-160 mg U/g dry cells with 100 ppm U in solution. The affinity of this biosorbent for the uranyl ion was found to be affected by timing of harvesting and medium composition. Uptake of the uranyl ion by nonliving biomass of Rhizopus oligosporus was due to ion exchange or complexation, since binding was reversed by the addition of complexing ligands or the reduction of pH to a value less than 2. Uptake isotherms were interpreted in terms of a model of multiple equilibria. At pH 相似文献   

Accumulation of uranium at low concentration by the green alga Scenedesmus obliquus 34

Accumulation of UO ₂ ^{2 +} by Scenedesmus obliquus 34 was rapid and energy-independent and the biosorption of UO ₂ ^{2 +} could be described by the Freundlich adsorption isotherm below the maximum adsorption capacity (75 mg g-¹ dry wt). The optimum pH for uranium uptake was between 5.0_8.5.0.1_2.0 M NaCl enhanced uranyl, while Cu²⁺, Ni²⁺, Zn²⁺, Cd²⁺ and Mn²⁺ competed slightly with uranyl. Pretreatment had an unexpected effect on biosorption. After being killed by 0.1 M HCl, S. Obliquus 34 showed 45% of the uptake capacity of the control in which fresh cells were suspended directly in uranyl solution, while the pretreatment of cells by 0.1 M NaOH, 2.0 M NaCl, ethanol or heating decreased uptake slightly. Fresh S. obliquus 34 at 1.2_2.4 mg dry wt mL-1 was able to decrease U from 5.0 to 0.05 mg L-1 after 4_6 equilibrium stages with batch adsorption. Deposited U could be desorbed by pH 4.0 buffer. It is suggested that U was captured by effective groups or by capillary action in the cell wall in the form of [UO2OH]+. This revised version was published online in August 2006 with corrections to the Cover Date.     

Sugar-induced potential difference and short circuit current in bullfrog small intestine: effect of UO22+.

UO22+ 1.3 mM added as UO2(NO3)2 to the mucosal solution consistently inhibited the P.D. and ISC evoked by 11 mM glucose and 35 mM 3-O-methyl glucose across isolated strips of bullfrog small intestine bathed by symmetrical Ringer solutions in which SO42- was the major anion. The average degree of inhibition in the presence of glucose was 42 +/- 7(SEM) percent. P.D. and ISC in the absence of transported solutes were not significatnly altered by mucosal UO22+ at this concentration. Increasing the mucosal UO22+ concentration to 2.6 mM did not significantly increase its inhibitory action on glucose-evoked P.D. and ISC. Further increasing the UO22+ concentration to 13 mM completely inhibited glucose-induced P.D. and ISC but also markedly reduced these parameters in the absence of glucose. Serosal UO22+ (1.3mM) had no effect on the P.D. and ISC evoked by glucose and 3-O-methyl glucose. It is suggested that the inhibitory action of UO22+ involves binding of this ion to anionic sites located in the apical membrane of the absorptive cells. Mucosal or serosal UO22+ (1.3 mM) had no effect on the P.D. and ISC elicited by 20 mM valine, indicating that under the conditions of these experiments UO22+ selectively inhibits sugar-induced P.D. and ISC and, by implication mucosal sugar uptake.     

Uranium(VI) complexes with phospholipid model compounds--a laser spectroscopic study

We present the complex formation of the uranyl ion (UO(2)(2+)) in the aqueous system with phosphocholine, O-phosphoethanolamine and O-phosphoserine. These phosphonates (R-O-PO(3)(2-)) represent the hydrophilic head groups of phospholipids. The complexation was investigated by time-resolved laser-induced fluorescence spectroscopy (TRLFS) at pH=2-6. An increase of the fluorescence intensity, connected with a strong red-shift of about 8 nm compared to the free uranyl ion, indicates a complex formation between UO(2)(2+) and the phosphonates already at pH=2. Even at pH=6 these complexes prevail over the uranyl hydroxide and carbonate species, which are generated naturally at this pH. At pH=4 and higher a 1:2 complex between uranyl and O-phosphoserine was found. Complexes with a metal-to-ligand ratio of 1:1 were observed for all other ligands. Fluorescence lifetimes, emission maxima and complex stability constants at T=22+/-1 degrees C are reported. The TRLFS spectra of uranyl complexes with two phosphatidic acids (1,2-dimyristoyl-sn-glycero-3-phosphate and 1,2-dipalmitoyl-sn-glycero-3-phosphate), which represent the apolaric site of phospholipids, show in each case two different species.     

钙、磷、镁和钾对雪莲细胞悬浮培养的影响

考察了钙、磷、镁和钾等大量营养元素对雪莲细胞TUIP-8悬浮培养的影响,确定了培养雪莲细胞的钙、磷、镁、钾的最佳浓度范围。低于以上4种大量营养元素的最适浓度范围,TUIP-8细胞的生长和黄酮合成都将明显受到抑制。该细胞对Ca^2 和Mg^2 具有较好的浓度耐受性,而对PO4^3-和K^ 的耐受性较差。     

Uranyl precipitation by Pseudomonas aeruginosa via controlled polyphosphate metabolism

The polyphosphate kinase gene from Pseudomonas aeruginosa was overexpressed in its native host, resulting in the accumulation of 100 times the polyphosphate seen with control strains. Degradation of this polyphosphate was induced by carbon starvation conditions, resulting in phosphate release into the medium. The mechanism of polyphosphate degradation is not clearly understood, but it appears to be associated with glycogen degradation. Upon suspension of the cells in 1 mM uranyl nitrate, nearly all polyphosphate that had accumulated was degraded within 48 h, resulting in the removal of nearly 80% of the uranyl ion and >95% of lesser-concentrated solutions. Electron microscopy, energy-dispersive X-ray spectroscopy, and time-resolved laser-induced fluorescence spectroscopy (TRLFS) suggest that this removal was due to the precipitation of uranyl phosphate at the cell membrane. TRLFS also indicated that uranyl was initially sorbed to the cell as uranyl hydroxide and was then precipitated as uranyl phosphate as phosphate was released from the cell. Lethal doses of radiation did not halt phosphate secretion from polyphosphate-filled cells under carbon starvation conditions.     

Uranyl Precipitation by Pseudomonas aeruginosa via Controlled Polyphosphate Metabolism

The polyphosphate kinase gene from Pseudomonas aeruginosa was overexpressed in its native host, resulting in the accumulation of 100 times the polyphosphate seen with control strains. Degradation of this polyphosphate was induced by carbon starvation conditions, resulting in phosphate release into the medium. The mechanism of polyphosphate degradation is not clearly understood, but it appears to be associated with glycogen degradation. Upon suspension of the cells in 1 mM uranyl nitrate, nearly all polyphosphate that had accumulated was degraded within 48 h, resulting in the removal of nearly 80% of the uranyl ion and >95% of lesser-concentrated solutions. Electron microscopy, energy-dispersive X-ray spectroscopy, and time-resolved laser-induced fluorescence spectroscopy (TRLFS) suggest that this removal was due to the precipitation of uranyl phosphate at the cell membrane. TRLFS also indicated that uranyl was initially sorbed to the cell as uranyl hydroxide and was then precipitated as uranyl phosphate as phosphate was released from the cell. Lethal doses of radiation did not halt phosphate secretion from polyphosphate-filled cells under carbon starvation conditions.     

On the mechanism of uranium binding to cell wall of <Emphasis Type="Italic">Chara fragilis</Emphasis>

Biosorption of uranium from nuclear waste liquids and contaminated surface waters and soils has recently attracted special interest. However, the detailed mechanism of uranium uptake by plants is not well understood. The aim of this work is to investigate the role of cell wall components of the freshwater alga Chara fragilis in uranium sequestration from its solution. Three types of algae preparations: extract of cell wall polysaccharides, dried and live algae were subjected to uranium solutions of different concentration and pH. FTIR and X-ray diffraction were used to assess both potential binding sites and the form of the uranyl sequestered by algae. Sorption of uranium by live and dry algae shows remarkable differences both in terms of overall uptake and mechanisms involved. All experiments are consistent with the conclusion that coprecipitation of uranyl species with CaCO3 is the major binding mechanism in uranium sequestration by Chara fragilis, while the direct exchange of Ca2+ with UO22+ has a minor role. Live algae are twice as efficient in sequestering uranium from solution than dried ones due to the formation of different crystalline forms such as aragonite and rutherfordine forming in live algae in the presence of the uranyl species in solution. It therefore appears that metabolic processes such as photosynthesis, most likely through the regulation of pH, play a key role in the uranium uptake by plants. Further understanding of the complex mechanism of metabolic control of the uranium uptake by plants is needed before the planning of bioremediation of this element.     

Bioaccumulation of nickel by microbially-enhanced chemisorption into polycrystalline hydrogen uranyl phosphate

Summary Ni²⁺ was removed quantitatively from aqueous solution into a microbially-created crystalline deposit of hydrogen uranyl phosphate (HUP). The mechanism of Ni²⁺ removal is an ion-exchange intercalation of Ni²⁺ into the interlayer space of HUP. The Ni²⁺-removing capacity of a column was proportional to the mass of HUP deposited and the Ni/HUP-loaded column was regenerated by washing with uranyl solution containing citrate/MOPS buffer and glycerol-2-phosphate, or with citrate buffer alone. Regeneration in the presence of UO₂ ²⁺ increased the Ni²⁺-removing capacity of the column. A new mechanism for the removal of heavy metals via microbially enhanced chemisorption is proposed.     

Purification of VP3 protein of infectious bursal disease virus using nickel ion-immobilized regenerated cellulose-based membranes

In this study, hexa-histidine tagged VP3 protein of infectious bursal disease virus (IBDV) was purified using immobilized metal ion affinity technique from the fermentation of Escherichia coli BL21 (DE3) containing a recombinant plasmid with a VP3 gene. The purification efficiencies of VP3 protein (TVP3 and DeltaTVP3) using Ni(2+)-NTA commercial agarose gels and Ni(2+)-IDA regenerated cellulose-based membranes at 4 degrees C were compared. A good washing condition for removing most impurity proteins was found as 20 mM NaH(2)PO(4), 500 mM NaCl, 40 mM imidazole, pH 7.8, whereas an efficient elution condition was 20 mM NaH(2)PO(4), 500 mM NaCl, 500 or 750 mM imidazole, pH 7.8. By applying these conditions to the flow experiments, similar recovery (86-88%) and purity (98-99%) for VP3 were obtained in both gel column (1 ml gel) and membrane cartridge (four membrane disks) under the flow rate of 1.7 ml/min for protein loading and 2.7 ml/min for protein elution. Regarding that the membrane process exhibited some advantages such as shorter residence time and lower cost, a better process efficiency in a large-scale system could be expected for the Ni(2+)-IDA membranes.     

High-performance analytical applications of immobilized metal ion affinity chromatography

The separation of three sets of standard protein mixtures on a high-performance immobilized metal ion affinity chromatography (HP-IMAC) column by elution with linear gradients of imidazole is described. The affinity of the test proteins for the immobilized metal ions follows the order Cu2+ greater than Ni2+ greater than Zn2+. The iminodiacetic acid-Cu2+ column gives the best resolution of all three protein mixtures and is the only immobilized metal ion column that can be used for elution of absorbed proteins with a decreasing pH gradient. An application of HP-IMAC for the separation of monoclonal IgG from mouse ascites fluid is also outlined. This versatile separation method is thus suitable for both analytical and preparative separations of proteins and peptides resulting in high recoveries and good reproducibility. The leakage of immobilized metal ions from the TSK gel chelate-5PW is apparent if the column is eluted by buffers containing low concentrations of (i) glycine or (ii) primary amines at round neutral pH. Considerable amounts of immobilized Zn2+ and Ni2+ ions also leak from the column by washing with buffers of pH 4.5 or lower. However, all three immobilized metal ions are stable toward exposure to low concentrations of imidazole (up to 50 mM) in phosphate buffers between pH 6.5 and 8.0. Adsorbed proteins could thus be eluted conveniently by using linear gradients of imidazole to give reproducible results. Moreover, this elution procedure made it possible to use the IMAC columns for repeated runs without the need for regeneration and recharging of the columns with fresh metal ions after each use.     

The Effects of Immobilization on Photosynthesis,Growth, Nitrogen Fixation and Ammonium Ion Excretion of Mutant and Wild Type Cells of Anabaena variabilis

Ammonium ion photoproduced by nitrogen-fixing cyanobacteria is formed from water, air nitrogen and solar energy under normal temperature and atmospheric pressure. It is reported here that the mutant and its parent wiid type cells of Anabaena variabilis from Shanmugan lab were immobilized into the polyurethane foams. The growth curves measured by cell counting, O.D750 measurement and ch]orophyll a determination showed that the growth of the immobilized wild type cells was more rapid than the immobilized mutant cells, and for both two types of cells the free-living cells grew better than the immobilized ones. The nitrogenase activity (reduction of C2H2) was 44% greater in immobilized mutant cells and 232% greater in immobilized wild type cells than both free-living cells, respectively. Free-living cells of wild type A. variabilis almost did not excrete any ammonium ion and the immobilized cells of the wild type had the ammonium ion excretion activity similar to that of the free-living mutant cells. Moreover, immobilization stimulated the activity of ammonium ion excretion 55.6% greater than the free-living cells, in the mutant strain. The fluorescence enhancement induced by DCMU (3-(3,4-dichlor-benzene)-1, 1-dimethl urea) indicated the photosynthetic capability of wild type ceils in both free-living and immobilized states were higher than that of the mutant cells, and the free-living cells of both two types of cells had higher capability than the immobilized cells. The fluorescence emission spectra at 77K showed that there were four peak emission: F646(F645 or F650) and F662(F664 or F665) from phycobilin, F693(F698,F700 or F702) from PS Ⅱ and F732(F728 or F733 from PS Ⅰ In both two types of immobilized cells the photosynthetic light energy distribution tended to be in PS Ⅰ and it seems to be benefitlal for the nitrogenase activity and ammonium ion excretion. SEM observation indicated that the shape and size of the immobilized cells remained unchanged incompared with the free-living cells, However, the surface of the immobilized cells had accumulated some mucilage, and particularly, the film of mucilage coated both surface of the mutant cells and the foam matrix.     

Microbially-enhanced chemisorption of Ni2+ ions into biologically-synthesised hydrogen uranyl phosphate (HUP) and selective recovery of concentrated Ni2+ using citrate or chloride ion

Hydrogen uranyl phosphate (HUP) deposited enzymatically on Citrobacter N14 immobilized in polyacrylamide gel removed nickel ions from solution via intercalative ion-exchange into the HUP lattice. Using flow-through columns containing 100 mg dry weight of biomass and 200–250 mg loaded uranium column saturation and breakthrough of Ni²⁺ occurred after ca. 600 ml, with a total of 30 mg Ni²⁺ loaded per column, corresponding to a molar ratio of U:Ni of 2:1, in accordance with the identity of the material as Ni(UO₂PO₄)₂, identified previously. Ni²⁺ was selectively desorbed using 100 mM sodium citrate-citric acid buffer over 140 ml or a short pulse (5 ml) of 500 mM citrate buffer followed by a water wash, giving a total recovery volume of 80 ml, with a total citrate concentration of 30 mM in the wash solution of the latter. As an alternative eluant which gives no residual BOD NaCl (0.6 M) or seawater gave comparable recovery of Ni²⁺ to the 0.5 M citrate pulse, but with a Ni²⁺ recovery volume of 40–50 ml. The concentration ratio of Ni²⁺-deposition:desorption (vol:vol) was 3–4 fold better with chloride ion than with 100 mM citrate.     

Binding of calcium and phosphate ions to dentin phosphophoryn

The concomitant binding of calcium and inorganic phosphate ions by the highly phosphorylated rat dentin phosphophoryn (HP) was measured in the pH range of 7.4-8.5 by an ultrafiltration procedure. HP binds almost exclusively the triply charged PO4(3-) ion, and for each PO4(3-) ion bound, the protein binds about 1.5 additional Ca2+ ions. Therefore, the protein-mineral ion complex can be described as a protein with two different ligands, Ca2+ ions and calcium phosphate clusters having a stoichiometry of about Ca1.5PO4. Empirically the binding of calcium and phosphate can best be described as a function of a neutral ion activity product in which 2.5-10% of the phosphate is HPO4(2-). The stoichiometry of the bound clusters is similar to that of amorphous calcium phosphate, and it is clear that the protein does not sequester crystal embryos of octacalcium phosphate or hydroxyapatite. The protein-mineral ion complex is amorphous by electron diffraction analysis and does not catalyze the formation of a crystalline phase when aged in contact with its solution. About 15% of the bound phosphate is buried in protected domains, and it is stable with respect to dissociation for extended periods in phosphate-free calcium buffers. The buried mineral maintains the protein in an aggregated state even at calcium ion concentrations which are too low for the aggregation of unmineralized HP. In vivo HP should be ineffective in the nucleation of a crystalline mineral phase, if it is secreted in a mineralized aggregated state similar to casein and the bivalve phosphoprotein.     

The use of Escherichia coli bearing a phoN gene for the removal of uranium and nickel from aqueous flows

A Citrobacter sp. originally isolated from metal-polluted soil accumulates heavy metals via metal-phosphate deposition utilizing inorganic phosphate liberated via PhoN phosphatase activity. Further strain development was limited by the non-transformability of this environmental isolate. Recombinant Escherichia coli DH5α bearing cloned phoN or the related phoC acquired metal-accumulating ability, which was compared with that of the Citrobacter sp. with respect to removal of uranyl ion (UO₂ ²⁺) from dilute aqueous flows and its deposition in the form of polycrystalline hydrogen uranyl phosphate (HUO₂PO₄). Subsequently, HUO₂PO₄-laden cells removed Ni²⁺ from dilute aqueous flows via intercalation of Ni²⁺ into the HUO₂PO₄ lattice. Despite comparable acid phosphatase activity in all three strains, the E. coli DH5α (phoN) construct was superior to Citrobacter N14 in both uranyl and nickel accumulation, while the E. coli DH5α (phoC) construct was greatly inferior in both respects. Expression of phosphatase activity alone is not the only factor that permits efficient and prolonged metal phosphate accumulation, and the data highlight possible differences in the PhoN and PhoC phosphatases, which are otherwise considered to be related in many respects. Received: 30 December 1997 / Received revision: 25 March 1998 / Accepted: 26 March 1998     

IDA型固定化镍离子金属螯合亲和膜色谱对人血清白蛋白的分离纯化

采用自制的固定化镍离子亚氨二乙酸(IDA)型复合纤维素金属螯合膜色谱对药用人血清白蛋白的进一步纯化进行了研究。考察了Ph对HAS吸附效果的影响。经一步纯化,商品药用HAS中的许多杂蛋白可被除去,经毛细管电泳分析,纯度与Sigma公司的电泳纯HAS相当,回收率可达85%以上。纯化蛋白液中的镍离子经过自制的N,N,N′-三羧甲基乙二胺(TED)型螯合柱处理后可较好地除去。