Silver ions induce a rapid Ca2+ release from isolated intact bovine rod outer segments by a cooperative mechanism

Summary Micromolar concentrations of silver ion activate large Ca²⁺ fluxes across the plasma membrane of intact rod outer segments isolated from bovine retinas (intact ROS). The rate of Ag⁺-induced Ca²⁺ efflux from intact ROS depended on the Ag⁺ concentration in a sigmoidal manner suggesting a cooperative mechanism with a Hill coefficient between 2 and 3. At a concentration of 50 m Ag⁺ the rate of Ca²⁺ efflux was 7×10⁶ Ca²⁺/outer segment/sec; this represents a change in total intracellular Ca²⁺ by 0.7mm/outer segment/sec. Addition of the nonselective ionophore gramicidin in the absence of external alkali cations greatly reduced the Ag⁺-induced Ca²⁺ efflux from intact ROS, apparently by enabling internal alkali cations to leak out. Adding back alkali cations to the external medium restored Ag⁺-induced Ca²⁺ efflux when gramicidin was present. In the presence of gramicidin, Ag⁺-induced Ca²⁺ efflux from intact ROS was blocked by 50 m tetracaine orl-cis diltiazem, whereas without gramicidin both blockers were ineffective. Bothl-cis diltiazem and tetracaine are blockers of one kinetic component of cGMP-induced Ca²⁺ flux across ROS disk membranes. The ion selectivity of the Ag⁺-induced pathway proved to be broad with little discrimination between the alkali cations Li⁺, Na⁺, K⁺, and Cs⁺ or between Ca²⁺ and Mg²⁺. The properties of the Ag⁺-induced pathway(s) suggest that it may reflect the cGMP-dependent conductance opened in the absence of cGMP by silver ions.     

Accumulation of silver by growing and non-growing populations of Citrobacter intermedius B6

Summary Accumulation of silver is reported for growing and non-growing populations of Citrobacter intermedius B6. In non-growing cultures a maximum uptake of 4.35% (w/w) was observed at an initial silver concentration of 2111.2 mol Ag⁺ l^-1. In contrast the maximum uptake of silver by growing bacteria was 2.81% (w/w) at an effective concentration of silver of 217.8 mol·l^-1. Silver accumulation rates in both resting (460 mol Ag⁺ g^-1 per hour) and continuously grown (41 mol Ag⁺ g^-1 per hour) bacteria are higher than previously reported. Cell fractionation and electron microscopy of continuously grown bacteria indicated that accumulation of silver was associated with the cell envelope, in the form of dense deposits of macromolecular proportions. This observation discounts simple surface adsorption as the process of accumulation in growing cultures.     

Protection of catheter surfaces from adhesion of Pseudomonas aeruginosa by a combination of silver ions and lectins

A catheter surface was modified by coating a cellulose acetate polymer. Adhesion of Pseudomonas aeruginosa ATCC 27853 to the surface was investigated by exposing bacterial cultures to three treatments: polymer impregnated with silver ions (Ag⁺), polymer surfaces coated with lectins and a combination of Ag⁺ and a lectin coating. The effective concentration of Ag⁺ providing protection against bacterial biofilm development was 100g/ml and higher. Lectins alone at 10% also showed inhibition of bacterial attachment. However, the best result was achieved against bacterial adhesion and growth on surfaces using a combination of 100 g Ag⁺/ml and a lectin coating as a surface treatment. This surface treatment was also effective against both fresh culture and a two-week-old culture containing P. aeruginosa producing exopolymers. Our results suggest that Ag⁺impregnation combined with a lectin coating warrants further investigation as a potential means of protecting catheters.     

Expression of lactate dehydrogenase isozyme 5 (LDH-5) in cultured mouse blastocysts in the absence of implantation and outgrowth

Extensive extraction studies with Triton X-100 revealed only LDH-1 (B₄) but no trace of LDH-5 (A₄) in one-cell and two-cell mouse and rat embryos. The LDH isozyme pattern of preimplantation mouse embryos changes from the maternally inherited B subunit isozyme (LDH-1) to a pattern dominated by LDH-5 when mouse blastocysts are cultured under conditions that prevent hatching but allow trophoblast giant cell transformation. During differentiation of mouse blastocysts in vitro, implantation is therefore not essential for the appearance of the A subunit form of LDH (LDH-5) coded for by the embryonic genome. Mechanisms controlling the expression of LDH-5 in mouse blastocysts during in vivo development are discussed.This work was supported by grants of the Deutsche Forschungsgemeinschaft awarded to the Sonderforschungsbereich 29—Embryonale Entwicklung and Differenzierung.     

Quenching of the fluorescence of proteins by silver nitrate

The mechanisms by which Ag⁺ may quench protein tryptophanyl fluorescence have been studied. A 1:1 Ag⁺-tryptophan complex was detected spectrophotometrically and shown to have a k_a = 6.5 × 10³ M^?1. The complex was nonfluorescent. Ag⁺ and NO₃^? each caused collisional quenching which proceeded at nearly diffusion-controlled rates in a series of indole-containing compounds. Analysis of the rates by means of Stern-Volmer plots and lifetime measurements showed also that charge and the presence of salt influence the quenching rate constants.The fluorescence of nonsulfhydryl proteins was quenched by AgNO₃ only in concentrations needed for Stern-Volmer quenching of simple indole model compounds. However, the plots for protein quenching were generally nonlinear, a reflection of the heterogeneity of tryptophanyl residues. AgNO₃ quenching increased the polarization of protein fluorescence and decreased the lifetime. Rotational relaxation times were determined from Perrin plots of reciprocal polarization vs fluorescence intensity in the presence of various amounts of AgNO₃.The fluorescence of the sulfhydryl proteins ovalbumin, yeast, and equine liver alcohol dehydrogenases was strongly quenched by AgNO₃ in parallel with the formation of Ag⁺-mercaptide bonds. The quenching of fluorescence of sulfhydryl proteins was exhibited even in 8 m urea, thus ruling out conformational change as a major basis for the quenching. It was found that Ag⁺ mercaptide bond formation was accompanied by development of an ultraviolet absorption band. The reaction of Ag⁺ with cysteine, for example, could be followed spectrophotometrically. The uv absorption of different silver mercaptides varied with the compound and pH.Since the uv absorption of Ag⁺-mercaptides extended up to 340 nm, and was also found in Ag⁺-treated sulfhydryl proteins, energy transfer from excited tryptophans seemed a reasonable basis for the observed fluorescence quenching. This possibility was confirmed by calculation of Förster critical transfer distances for a variety of donor-acceptor (Ag⁺-mercaptide) pairs.The lifetime of sulfhydryl protein fluorescence was decreased by AgNO₃, but the emission spectrum was relatively little affected, in contrast to previously reported quenching by Hg²⁺. Additional mechanisms of fluorescence alteration by Ag⁺ in proteins (e.g., “heavy atom” effect, conformational changes, enhancement of sulfhydryl quenching) are also considered.The spectral effects of Ag⁺ interaction with proteins have the following practical applications:determination of —SH groups; probe of accessibility of binding sites and tryptophan-sulfhydryl distances; determination of rotational relaxation times by Perrin plots of reciprocal polarization vs lifetime; kinetic studies of Ag⁺ interaction with proteins.     

The effects of silver ions and silver nanoparticles on cell division and expression of <Emphasis Type="Italic">cdc2</Emphasis> gene in <Emphasis Type="Italic">Allium cepa</Emphasis> root tips

The effects of silver nanoparticles (AgNPs), silver ions (Ag⁺), and polyvinylpyrrolidone (PVP) on mitosis and expression of a gene encoding cyclin-dependent kinase 2 (cdc2) in onion roots were compared. Three concentrations (5, 10, and 15 mg dm^-3) were employed in combination with three incubation times (3, 6, and 9 h). PVP enhanced mitotic index and cdc2 expression. Both silver forms decreased mitotic index and cdc2 expression. Genotoxicity of both silver forms were indicated by three major distinguishable classes of chromosome aberrations: spindle disturbances, clastogenic aberrations, and chromosome stickiness. Concerning Ag⁺ treatments, significant enhancements in occurrence of any chromosome aberration type was associated with significant decrease in mitotic index. On the other hand, disturbed spindle in AgNPs treatments was observed even in absence of significant reduction in mitotic index suggesting that AgNPs inhibit cellular events occurring during mitosis to proceed normally rather than starting of cell division.     

Enzymatic relationship between human Tγ+ lymphocytes and thymocytes

The lactate dehydrogenase isoenzyme pattern has been determined in human thymocytes and in peripheral blood T lymphocytes, Tγ⁺, and Tμ⁺ lymphocytes. Thymocytes have a highly significant lower activity in LDH-1 and LDH-2 together with a highly significant higher activity in LDH-3 and LDH-4 than peripheral T lymphocytes. The same differences are seen when Tγ⁺ and Tμ⁺ cells are compared. On the contrary there are almost no differences in pattern between the peripheral T lymphocytes and Tμ⁺ cells on one hand and between thymocytes and Tγ⁺ cells on the other hand. These findings suggest that both thymocytes and Tγ⁺ cells exhibit a very immature LDH pattern with regard to the Tμ⁺ cell population.     

Efficient disinfection of Escherichia coli in water by silver loaded alumina

The catalytic inactivation of Escherichia coli (E. coli) in water by silver loaded alumina as catalyst was investigated. Ag/Al₂O₃ and AgCl/Al₂O₃ catalysts exhibited high bactericidal activity at room temperature in water with no need for any light or electrical power input. Dissolved oxygen which can be catalyzed to reactive oxygen species (ROS) was found to be essential for the strong bactericidal activities of the catalysts. Decomposition of the cell wall leading to leakage of the intracellular component and the complete lysis of the whole cell were directly observed by transmission electron microscopy (TEM). The resultant change in cell permeability was confirmed by potassium ion leakage. The different morphological changes between E. coli cells treated with the catalysts and Ag⁺ were also observed. The formation of ROS involved in the bactericidal process by AgCl/Al₂O₃ was confirmed by addition of catalase and OH scavenger. Higher temperature and pH value were found to have positive effect on the bactericidal activity of AgCl/Al₂O₃. All these results indicated that the bactericidal effect of the catalyst was a synergic action of ROS and Ag⁺, not an additive one. A possible mechanism is proposed.     

The self-association of jack bean urease and its modification by silver ions.

At low ionic strength urease has been found to dissociate at protein concentrations below 1 × 10⁸m. The inhibition of enzyme activity by Ag⁺ has been used to demonstrate this. The inhibition by Ag⁺ has been shown to be independent of dissociation but, at dilutions where dissociation occurs, silver ion modifies the process. Urease is aggregated by Ag⁺ at high Ag⁺:protein ratios. Such inactive aggregates can be solubilized and reactivated by dithiothreitol. Further evidence has been obtained indicating the similarity of the (8n) and (16n) forms of urease. The phenomena of inhibition and aggregation in the presence of the heavy metal ion have been shown to be separate processes.     

Amicyanin metal-site structure and interaction with MADH: PAC and NMR spectroscopy of Ag-, Cd-, and Cu-amicyanin

To investigate the structural control mechanisms in the metal site of amicyanin when interacting with MADH, redox-inactive Ag⁺- and Cd²⁺-substituted amicyanins were studied with perturbed angular correlations of -rays (PAC) spectroscopy. PAC experiments on ^111mCd-substituted amicyanin revealed two different metal-site structures, which are very likely in dynamic exchange on a ~5 ns timescale. Only one structure binds to MADH. The dissociation constants, K _d, are 9±2 M with MADH^red and 38±11 M with MADH^ox, indicating that the Cd-amicyanin binding affinity is regulated by the MADH redox state. PAC experiments on ¹¹¹Ag-substituted amicyanin also showed two different forms of Ag-amicyanin, probably reflecting relaxation from Ag to Cd geometry. No binding of Ag-amicyanin to MADH could be observed with PAC, suggesting that the K _d is larger than 43 M, based on the 95% confidence limit. NMR revealed large chemical shift differences between native copper amicyanin and both metal-substituted forms. Affected residues are found up to 15 Å away from the metal ion. The Ag⁺- and Cd²⁺-substituted amicyanins demonstrate no change in coordination as a function of pH, contrary to Cu⁺-amicyanin which shows protonation of the copper ligand His96 with pK _a=6.8. It is concluded that, contrary to other blue copper proteins, Ag⁺-amicyanin is not a close mimic of Cu⁺-amicyanin, and that structural changes in the metal site have large effects on the affinity for the redox partner.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations AOM angular overlap model - HSQC heteronuclear single-quantum coherence - MADH methylamine dehydrogenase - MADH^ox oxidized MADH - MADH^red reduced MADH - NOESY nuclear Overhauser effect spectroscopy - NQI nuclear quadrupole interaction - PAC perturbed angular correlations of -rays - TOCSY total correlation spectroscopy     

A method to increase silver biosorption by an industrial strain of Saccharomyces cerevisiae

 Ag⁺ biosorption by an industrial strain of Saccharomyces cerevisiae was investigated. Older (96 h old) biomass had half the biosorption capacity of younger (24 h old) biomass (0.187 and 0.387 mmol Ag⁺/g dry mass respectively). Comparisons of cell walls isolated from biomass of either age indicated that chemical composition and Ag⁺ biosorption capacity varied little over the time span examined and that cell walls from either age of culture had small Ag⁺ biosorption capacities compared to whole cells of a similar age. Silver-containing precipitates were observed both on the cell wall and within the cell, indicating that intracellular components sorbed Ag⁺. The concentration of these precipitates within the cell appeared visually to decrease with age in Ag⁺-exposed cells. Incorporation of L-cysteine into the growth medium resulted in biomass with increased silver biosorption capacities, protein and sulphydryl group content. Increasing the concentration of L-cysteine in the growth medium from 0 to 5.0 mM increased silver biosorption from 0.389 to 0.556 mmol Ag⁺/g dry mass Isolated cell walls of biomass grown in supplemented media also showed a possible link between silver biosorption capacities, protein and sulphydryl group content. No precipitates were observed in silver-exposed biomass that had been grown in the presence of 5.0 mM L-cysteine. Received: 12 April 1995 / Received revision: 7 August 1995 / Accepted: 22 August 1995     

Bioaccumulation of silver by a multispecies community of bacteria

A stable community of bacteria that had unusually high tolerance of soluble silver was isolated from soil by chemostat enrichment. The community consisted of three bacteria: Pseudomonas maltophilia, Staphylococcus aureus and a coryneform organism. The pseudomonas was primarly responsible for the silver resistance. The tolerance of high silver concentrations, up to 100 mM Ag⁺, was greatly reduced when the community was grown in the absence of silver. Pseudomonas maltophilia comprised approximately 50% by numbers of the community when grown in chemostats in the presence or absence of Ag⁺ but large fluctuations occurred in population sizes of the other two bacteria; the S. aureus population was small (less than 1%) in the presence of Ag⁺ but comparised a third of the total numbers when Ag⁺ was omitted from the medium. Silver-resistant respiration of the silveradapted community was significant even when it was confronted with high concentrations of Ag⁺. In contrast the respiration of the coryneform organism and particularly S. aureus was highly sensitive to silver. The inhibition constants for silver-sensitive respiration were 0.78 mM and 0.04 mM for silver acclimatized and nonacclimatized communities respectively.The community had great capacity for silver bioaccumulation. Maximum concentrations of over 300 mg silver per g dry weight of biomass were recorded at an accumulation rate of 21 mg Ag⁺ h^-1 (g biomass)^-1. The extent of silver removal from solution was a function of initial concentration of silver; at low external concentrations (ca. 1 mM) all the silver was rapidly removed from solution, at high concentrations (ca. 12 mM) 84% removal occurred in 15 h.     

Functional and Biochemical Characterization of Cucumber Genes Encoding Two Copper ATPases CsHMA5.1 and CsHMA5.2

Plant copper P_1B-type ATPases appear to be crucial for maintaining copper homeostasis within plant cells, but until now they have been studied mostly in model plant systems. Here, we present the molecular and biochemical characterization of two cucumber copper ATPases, CsHMA5.1 and CsHMA5.2, indicating a different function for HMA5-like proteins in different plants. When expressed in yeast, CsHMA5.1 and CsHMA5.2 localize to the vacuolar membrane and are activated by monovalent copper or silver ions and cysteine, showing different affinities to Cu⁺ (K_m ∼1 or 0.5 μm, respectively) and similar affinity to Ag⁺ (K_m ∼2.5 μm). Both proteins restore the growth of yeast mutants sensitive to copper excess and silver through intracellular copper sequestration, indicating that they contribute to copper and silver detoxification. Immunoblotting with specific antibodies revealed the presence of CsHMA5.1 and CsHMA5.2 in the tonoplast of cucumber cells. Interestingly, the root-specific CsHMA5.1 was not affected by copper stress, whereas the widely expressed CsHMA5.2 was up-regulated or down-regulated in roots upon copper excess or deficiency, respectively. The copper-induced increase in tonoplast CsHMA5.2 is consistent with the increased activity of ATP-dependent copper transport into tonoplast vesicles isolated from roots of plants grown under copper excess. These data identify CsHMA5.1 and CsHMA5.2 as high affinity Cu⁺ transporters and suggest that CsHMA5.2 is responsible for the increased sequestration of copper in vacuoles of cucumber root cells under copper excess.     

Experimental evolution of Pseudomonas putida under silver ion versus nanoparticle stress

Whether the antibacterial properties of silver nanoparticles (AgNPs) are simply due to the release of silver ions (Ag⁺) or, additionally, nanoparticle-specific effects, is not clear. We used experimental evolution of the model environmental bacterium Pseudomonas putida to ask whether bacteria respond differently to Ag⁺ or AgNP treatment. We pre-evolved five cultures of strain KT2440 for 70 days without Ag to reduce confounding adaptations before dividing the fittest pre-evolved culture into five cultures each, evolving in the presence of low concentrations of Ag⁺, well-defined AgNPs or Ag-free controls for a further 75 days. The mutations in the Ag⁺ or AgNP evolved populations displayed different patterns that were statistically significant. The non-synonymous mutations in AgNP-treated populations were mostly associated with cell surface proteins, including cytoskeletal membrane protein (FtsZ), membrane sensor and regulator (EnvZ and GacS) and periplasmic protein (PP_2758). In contrast, Ag⁺ treatment was selected for mutations linked to cytoplasmic proteins, including metal ion transporter (TauB) and those with metal-binding domains (ThiL and PP_2397). These results suggest the existence of AgNP-specific effects, either caused by sustained delivery of Ag⁺ from AgNP dissolution, more proximate delivery from cell-surface bound AgNPs, or by direct AgNP action on the cell's outer membrane.     

Biosynthesis of silver nanoparticles using Bacillus subtilis EWP-46 cell-free extract and evaluation of its antibacterial activity

This study highlights the ability of nitrate-reducing Bacillus subtilis EWP-46 cell-free extract used for preparation of silver nanoparticles (AgNPs) by reduction of silver ions into nano silver. The production of AgNPs was optimized with several parameters such as hydrogen ion concentration, temperature, silver ion (Ag⁺ ion) and time. The maximum AgNPs production was achieved at pH 10.0, temperature 60 °C, 1.0 mM Ag⁺ ion and 720 min. The UV–Vis spectrum showed surface plasmon resonance peak at 420 nm, energy-dispersive X-ray spectroscopy (SEM–EDX) spectra showed the presence of element silver in pure form. Atomic force microscopy (AFM) and transmission electron microscopy images illustrated the nanoparticle size, shape, and average particle size ranging from 10 to 20 nm. Fourier transform infrared spectroscopy provided the evidence for the presence of biomolecules responsible for the reduction of silver ion, and X-ray diffraction analysis confirmed that the obtained nanoparticles were in crystalline form. SDS-PAGE was performed to identify the proteins and its molecular mass in the purified nitrate reductase from the cell-free extract. In addition, the minimum inhibitory concentration and minimum bactericidal concentration of AgNPs were investigated against gram-negative (Pseudomonas fluorescens) and gram-positive (Staphylococcus aureus) bacteria.     

The effects of salinity on ion concentrations within the root cells of Zea mays L.

Zea mays is a salt-sensitive crop species which in saline (100 mol m^-3 NaCl) conditions suffers considerable growth reduction correlated with elevated Na⁺ and Cl^- concentration within the leaves. To increase understanding of the regulation of ion uptake and transport by the roots in saline conditions, ion concentrations within individual root cortical cells were determined by X-ray microanalysis. There was variation in Na⁺, K⁺ and Cl^- distributions among individual cells, which could not be correlated with their spatial position in the roots. Generally, however, in response to saline growth conditions (100 mol m³ NaCl) Na⁺ and Cl^- were mostly localized in the vacuoles, although their concentrations were also sometimes increased in the cytoplasm and cell walls. The concentration of K⁺ in the cytoplasm was usually maintained at a level (mean 79 mol m^-3) compatible with the biochemical functions ascribed to this ion.Abbreviation (T)AEM (Transmission) analytical electron microscopy     

Forage Crop <Emphasis Type="Italic">Lolium multiflorum</Emphasis> Assisted Synthesis of AgNPs and Their Bioactivities Against Poultry Pathogenic Bacteria in In Vitro

Italian ryegrass is one of main feed for livestock animals/birds. It has potential antioxidant metabolites that can improve their health and protect them against various infectious diseases. In this work, we studied synthesis of silver nanoparticles assisted by forage crop Lolium multiflorum as a green synthesis way. Potential antibacterial efficacy of these synthesized nanosized silver nanoparticles against poultry pathogenic bacteria was then studied. Aqueous extract of IRG was used as reducing agent for bio-reduction of silver salt to convert Ag⁺ to Ag⁰ metallic nano-silver. Size, shape, metallic composition, functional group, and crystalline nature of these synthesized silver nanoparticles were then characterized using UV–Vis spectrophotometer, FESEM, EDX, FT-IT, and XRD, respectively. In addition, antibacterial effects of these synthesized AgNPs against poultry pathogenic bacteria were evaluated by agar well diffusion method. UV–Vis spectra showed strong absorption peak of 440–450 nm with differ reaction time ranging from 30 min to 24 h. FESEM measurements revealed particles sizes of around 20–100 nm, majority of which were spherical in shape while a few were irregular. These biosynthesized silver nanoparticles using IRG extract exhibited strong antibacterial activities against poultry pathogenic microorganisms, including Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli, and Bacillus subtilis. Overall results confirmed that IRG plant extract possessed potential bioactive compounds for converting silver ions into nanosized silver at room temperature without needing any external chemical for redox reaction. In addition, such synthesized AgNPs showed strong antibacterial activities against pathogenic bacteria responsible for infectious diseases in poultry.     

Antifungal and physical characteristics of modified denture base acrylic incorporated with silver nanoparticles

doi: 10.1111/j.1741‐2358.2011.00489.x Antifungal and physical characteristics of modified denture base acrylic incorporated with silver nanoparticles Objective: This study evaluated the antifungal and physical characteristics of denture base acrylic combined with silver nanoparticles. Materials and methods: Polymerized denture acrylic disc specimens containing 0 (control), 1.0, 5.0, 10.0, 20.0 and 30.0 wt% of silver nanoparticles were placed on separate culture plate dish and 100 ìL samples of yeast suspension of Candida albicans strain were inoculated on each specimens and incubated at 37°C, for 24 h. The antifungal effects were evaluated as a number of viable cells in retrieved fungal suspension. To characterize physical aspects, specimens were tested for elution of silver cation (Ag⁺) at 24 h and 30th day, thermal analysis (TG/DTA), scanning electron microscope and energy dispersed X‐ray analysis (SEM/EDX) and color stability. Results: Significant reduced CFU was exhibited at 20.0 and 30.0 wt% of silver nanoparticles incorporated (p < 0.01) and Ag⁺ elution from specimens (maximum 0.356 ± 0.11 mg/L) contributed little to the antifungal activity considering MIC of Ag⁺ in this study (3.0 mg/L). The successful synthesis of modified denture acrylic containing silver nanoparticles was accessed by TG/DTA and EDX analysis. Conclusion: The modified denture base acrylic combined with silver nanoparticles displayed antifungal properties and acted like latent antifungal material itself with low‐releasing Ag⁺, however, the improvement of poor color stability was still required.     

Biosorption of silver ions by processed Aspergillus niger biomass

Summary An alkali treated A. niger biomass was found to efficiently sequester silver ions from dilute as well as concentrated solutions (2.5–1000 ppm Ag⁺), with an ability to bind it to a level of upto 10% of dry weight. Biosorption of silver ions was not influenced by pH between 5–7. The bound Ag⁺ could be fully desorbed by dilute HNO₃ and the biosorbent regenerated by washing with Ca²⁺/Mg²⁺ solution. This biosorbent is unique in that the mechanism of metal ion sorption has been found to be exclusively by stoichiometric exchange with Ca²⁺ and Mg²⁺ of the biosorbent.