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1、毕业设计（论文）外文文献翻译院 系：厦门理工学院 环境工程年级专业：姓 名：学 号：附 件：Bacterial Sorption of Heavy Metals指导老师评语： 指导教师签名：年 月 日Bacterial Sorption of Heavy MetalsFour bacteria, Bacillus cereus, B.subtilis, Escherichia coli, and Pseudomonas aeruginosa, were examined for the ability to remove Ag+, Cd2+, Cu2+, and La3+ from solut
2、ion by batch equilibration methods. Cd and Cu sorption over the concentration range 0.001 to 1 mM was described by Freundlich isotherms. At 1 mM concentrations of both Cd2+ and Cu2+, P. aeruginosa and B. cereus were the most and least efficient at metal removal, respectively. Freundlich K constants
3、indicated that E. coli was most efficient at Cd2+ removal and B. subtilis removed the most Cu2+. Removal of Ag+ from solution by bacteria was very efficient; an average of 89% of the total Ag+ was removed from the 1 mM solution, while only 12, 29, and 27% of the total Cd2+, Cu2+, and La3+, respectiv
4、ely, were sorbed from 1 mM solutions. Electron microscopy indicated that La3+ accumulated at the cell surface as needlelike, crystalline precipitates. Silver precipitated as discrete colloidal aggregates at the cell surface and occasionally in the cytoplasm. Neither Cd2+ nor Cu2+ provided enough ele
5、ctron scattering to identify the location of sorption. The affinity series for bacterial removal of these metals decreased in the order Ag La Cu Cd. The results indicate that bacterial cells are capable of binding large quantities of different metals. Adsorption equations may be useful for describin
6、g bacterium-metal interactions with metals such as Cd and Cu; however, this approach may not be adequate when precipitation of metals occurs.The fate of toxic metallic cations in the soil environment depends largely on the interactions of these metals with inorganic and organic surfaces. The extent
7、to which a metallic cation interacts with these surfaces determines the concentration of metal in solution and, consequently, the potential for movement into groundwater or uptake by plants. A considerable amount of work has been done to evaluate the adsorption or complexation of various heavy metal
8、s by soils (11) and soil constituents, such as clays (22) and organic matter fractions (28). One potentially important organic surface which has received little attention is that of the soil microbial population. Soil microorganisms are typically associated with the clay and organic fractions of the
9、 soil microenvironment (21) and would be expected to participate in the metal dynamics typically ascribed to these fractions. Bacteria have a high surface area-to-volume ratio (2) and, as a strictly physical cellular interface, should have a high capacity for sorbing metals from solution. There is e
10、vidence that bacterial cells are more efficient at metal removal than clay minerals on a dry-weight basis (31). Kurek and co-workers (17) observed that sorption of Cd2+ by dead cells of a Paracoccus sp. and Serratia marcescens was greater than that of montmorillonite when the solid-to- solution rati
11、o was the same for both bacteria and clay. Live cells accumulated about the same quantity of Cd2+ as did clay.Several investigations have shown that relatively large quantities of metallic cations are complexed by algae (19), bacteria (29), and fungi (20). Metal binding by isolated gram-positive and
12、 gram-negative bacterial cell walls has also been evaluated (3, 5, 6, 10, 20). Cell walls of the gram-positive bacteria Bacillus subtilis and B.licheniformis were observed to bind larger quantities of several metals than cell envelopes of the gram-negative bacterium Escherichia coli(3).We are intere
13、sted in the role of microorganisms in the behavior of various heavy metals in the soil environment. The objectives of this work were to determine the metal-binding capacities of whole cells of two gram-positive and two gram-negative bacteria and to determine whether an equilibrium model, the Freundl
14、ich adsorption isotherm, would adequately describe bacterial metal sorption. B.cereus, B.subtilis, and Pseudomonas aeruginosa were examined as representatives of common species frequently isolated from soils. E.coli was also used as a second gram-negative bacterium because it is a well-characterized
15、 microorganism and its cell envelope has been shown to bind less metal than do B.subtilis cell walls (3). The four metallic ions used in this investigation were Ag+, Cd2+, Cu2+, and La3+. Cadmium and copper are both toxic cations of environmental importance. Silver and lanthanum, representative of m
16、onovalent and trivalent heavy metals, respectively, are also toxic but are less frequently found in the environment.MATERIALS AND METHODSBacteria and growth conditions. The bacteria used in these experiments were B. cereus ATCC 11778; P. aeruginosa ATCC 14886, both obtained from the American Type Cu
17、lture Collection; B. subtilis 168; and E. coli K-12 strain AB264, both from the University of Guelph. The bacteria were routinely cultured in 0.5x brain heart infusion broth (BBL Microbiology Systems) amended with 2.4 g of HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) liter-1 and 2.0 g
18、of MES (2-N-morpholinoethanesulfonic acid) liter-1 to buffer the medium acidity. The medium acidity was adjusted to pH 6.8 with 0.5 M KOH. Two 2-milliliter samples of late-exponential-phase starter cultures were used to inoculate 800 ml of broth in 3-liter Erlenmeyer flasks. Cells were grown to the
19、late-exponential phase at room temperature (approximately 23C) on an orbital shaker at 150 rpm. Cells of B. cereus grown in this manner remained in the vegetative state. The cells were harvested by centrifugation and washed twice with cold 10 mM Ca(NO3)2 which had been adjusted to pH 4.0 with 0.5 M
20、HNO3. The washed cells were resuspended in the Ca(NO3)2 solution at a concentration of approximately 12 mg (dry weight) ml-1 and stored for 2 to 4h at 5C before use. The Ca(NO3)2 solution was also used to make up all of the metal solutions and acted as an ionic strength buffer. Metal sorption studie
21、s. The four metal salts used in this study were AgNO3，Cd(NO3)24H2O，Cu(NO3)22.5H2O and La(NO3)26H2O. The metal solutions were adjusted to pH 4.0 with 0.5 M HNO3 to avoid precipitation of Cu as CuCO3. Identification of these metals in solution at pH 4.0 was done with the GEOCHEM computer program (27).
22、 At pH 4.0 in a Ca(NO3)2 matrix, these metallic cations were predicted to be found primarily in the free ionic form (97% in all cases).All of the plasticware used in these studies was leached in 3 M HNO3 and rinsed several times with double-deionized water before use to avoid metal contamination. A
23、batch equilibration method was used to determine sorption of metals by bacteria. Two milliliters of washed cells was placed in a 10-ml polypropylene centrifuge tube containing 6 ml of cold 10 mM Ca(NO3)2; and 1 ml of metal stock solution was added. The tubes were capped, placed on an inverting shake
24、r, and equilibrated for 2 h at 5C. After 2 h, the cells were removed from solution by centrifugation and the supernatant was collected and used for metal analysis. Equilibrium metal concentrations were determined by inductively coupled argon plasma spectroscopy on a Thermo Jarrell-Ash Plasma 300 spe
25、ctrometer. The amount of metal removed by the cells was determined on a dry-weight basis. To determine sorption isotherms, final metal concentrations for Cd2+ were 1, 0.1, 0.01, and 0.001 mM. Initial experiments indicated that sorption of Cu2+ from the 0.001 Mm treatment resulted in equilibrium conc
26、entrations below the detection limit. Subsequently, the most dilute concentration of Cu2+ used was 0.005 mM. Sorption of Ag+ and La3+ from 0.01 and 0.001 mM solutions also typically resulted in concentrations below the detection limit. The concentrations of Ag and La evaluated were 10, 1, 0.1, and 0
27、.01 mM.The sorption experiment was set up as an unbalanced four-by-four lattice with three replications over time (8). Each block within replicates contained four different bacterium-metal combinations at all four metal concentrations. When appropriate, the sorption isotherms were constructed by the
28、 methods outlined by Dao et al. (9) with the GLM procedure of the SAS statistical program (25). Electron microscopy. Electron microscopy was done to visualize the location of metals on the bacterial cells. Cells were equilibrated with 1 mM metal solutions as described above and fixed for 30 min at r
29、oom temperature in 5% glutaraldehyde (EM grade; Polysciences Inc.) containing the metal of interest at a concentration approximately equal to the equilibration concentration. The cells were then washed free of glutaraldehyde with the metal solution, enrobed in 2% Noble agar (Difco Laboratories), deh
30、ydrated through an ethanol-propylene oxide series, and embedded in SPURR (Polysciences Inc.). The embedded cells were thin sectioned on a Reichert Ultracut E ultramicrotome, and sections were collected on Formvar carbon-coated 200-mesh copper or aluminum grids. Sections of metal-treated cells were n
31、ot stained; the electron scattering provided by the sorbed metals acted as a contrasting agent (5). Some control cells were lightly stained with 2% uranyl acetate for 2 min to provide better visualization of these cells. Electron micros-copy was performed at 100 kV on a Philips EM-400T equipped with
32、 an EDAX energy-dispersive X-ray spectrometer interfaced with a Tracor Northern multichannel analyzer. Energy-dispersive X-ray analysis was used to confirm the identities of metals on the cells. Affinity series determination. To further elucidate the affinity of the bacterial cells for these metals,
33、 the cells were equilibrated for 2 h at 5C in solutions containing either the single metal at an initial concentration of 1 mM or all four metals at 1 mM each. As described above, all metal solutions were made in pH 4.0 10 mM Ca(NO3)2. After the cells were harvested by centrifugation, metals in the
34、supernatant were determined by inductively coupled argon plasma spectroscopy. The experiment was replicated three times. Data were subjected to analysis of variance procedures of the SAS statistical program (25), and means were separated by the least-significant-difference method. RESULTS Bacterial
35、sorption of Cd2+ and Cu2+ from solution was described well by the linearized Freundlich adsorption isotherm equation, log10S = log1OK + nlog1OC,where S is the amount of metal adsorbed in micromoles per gram, C is the equilibrium solution concentration in micromoles per liter, and K and n are Freundl
36、ich constants. The Freundlich constants for Cd2+ and Cu2+ sorption by the four bacteria are given in Table 1. The constant K represents the predicted quantity of metal removed in micromoles of metal per gram of dry cells at an equilibrium concentration of 1 uM, and n is the slope of the isotherm. Ex
37、amination of K values for Cd sorption showed that the gram-negative bacterium E. coli was most efficient at Cd sorption and P. aeruginosa also tended to sorb more Cd2+ than did the gram-positive bacteria. B. subtilis removed the most Cu2+ at an equilibrium concentration of 1 uM. However, only a 1.9-
38、fold difference in Cu sorption was observed between the most and least efficient bacteria, B. subtilis and B. cereus, respectively. Representative plots of adsorption isotherms for Cd2+ and Cu2+ are shown in Fig. 1. Analysis of covariance indicated that the slopes of the isotherms were different for
39、 the four bacteria; at high equilibrium concentrations, P.aeruginosa and B. cereus were most and least efficient, respectively, at removing Cd2+ and Cu2+ from solution. Freundlich isotherms were not useful for describing the removal of Ag+ and La3+ from solution because there were too few datum poin
40、ts over a wide range of concentrations. Equilibrium concentrations of La3+ were below detection limits when the initial concentration was 10 uM. Several of the observations for Ag+ equilibrium concentrations were also below detection limits at the 10 uM concentration. When the equilibrium concentrat
41、ion was below detection limits, the total metal bound on a dry-weight basis was calculated with the assumption that essentially all of the Ag+ or La3+ in solution was bound. A 10 mM treatment was included for these metals to extend the concentration range examined. A good relationship was found for
42、removal of Ag+ from solution as a function of the initial Ag+ concentration from 10 to 1,000 p.M (Fig. 2). There were no significant differences in Ag+ removal among bacteria. Saturation of the cells with Ag+ apparently occurred in the 10 mM Ag+ treatment, as the total Ag+ bound increased only about
43、 2-fold over a 10-fold increase in Ag+ concentration.Significant differences among the bacteria for La3+ removal were found in the 1 mM treatment when 33, 70, 114, and 144 p.mol g-1 were removed by B.cereus, E. coli, B.subtilis, P.aeruginosa, respectively (Fig.3). There were no significant differenc
44、es among bacteria for La3+ removal from the 10 or 100 uM solutions. The bacterial cells were evidently saturated with La at the 1 mM concentration, as very little additional La3+ was bound by these cells from the 10 mM La3+ treatment (Fig. 3). Silver was removed from solution much more efficiently t
45、han were the other metals at the 1 and 0.1 mM concentrations (Table 2). An average of 99% of the total Ag+ was removed from solution in the 0.1 mM treatment. Cadmium was bound by the cells to a much lesser extent, with only 12 and 23% of the total Cd2+ removed from the 1 and 0.1 mM treatments, respe
46、ctively. Even in the 0.001 mM treatment, an average of 46% of the added Cd2+ remained in solution (data not shown).Electron micrographs of metal-treated cells showed that Ag was associated with the cell primarily as discrete particles at or near the cell walls of the bacteria, whether they were gram
47、 positive or gram negative (Fig. 4). Energy-dispersive X-ray analysis confirmed that the particles were silver. Attempts were also made to identify the form of silver in the particles by using select-area electron diffraction; however, the particles were too small to produce useful transforms. Lanth
48、anum was also easily observed on the cells in thin sections. The bound La was observed as needlelike precipitates deposited uniformly around the cell wall periphery (Fig.5). Energy-dispersive X-ray analysis confirmed that the bound metal was La, and select-area electron diffraction analysis indicate
49、d that the precipitate was crystalline. There was no evidence of uniform dispersal of either Ag or La in the cytoplasm, indicative of energized uptake. However, discrete Ag particles were occasionally found in the cytoplasm (ca. 1% of the cells), generally near the cell plasma membrane, and it is possible that these cells represent nonviable bacteria within the population.Neither Cu2+ nor Cd2+ provided eno
