Removal of heavy metals using a brewer's yeast strain of Saccharomyces cerevisiae: advantages of using dead biomass

Aim:  The capacities of live and heat-killed cells of Saccharomyces cerevisiae at 45°C for the removal of copper, nickel and zinc from the solution were compared.
Methods and Results:  Kinetic studies have shown a maximum accumulation of Ni²⁺ and Zn²⁺ after 10 min for both types of cells, while for Cu²⁺ this was attained after 30 and 60 min for dead and live cells, respectively. Equilibrium studies have shown that inactivated biomass displayed a greater Zn²⁺ and Ni²⁺ accumulation than live yeasts. For Cu²⁺, live and dead cells showed similar accumulation. Fluorescence, scanning electron microscopy and infrared spectroscopy studies have shown that no appreciable structural or molecular changes occurred in the cells during the killing process. The increased metal uptake observed in dead cells can be most likely explained by the loss of membrane integrity, which allows the exposition of further metal-binding sites present inside the cells.
Conclusions:  Heat-killed cells showed a higher degree of heavy metal removal than live cells, being more suitable for further bioremediation works.
Significance and Impact of the Study:  Dead flocculent cells can be used in a low cost technology for detoxifying metal-bearing effluents as this approach combines an efficient metal removal with the ease of cell separation.     

Mechanisms of strontium uptake by laboratory and brewing strains of Saccharomyces cerevisiae.

Laboratory and brewing strains of Saccharomyces cerevisiae were compared for metabolism-independent and -dependent Sr2+ uptake. Cell surface adsorption of Sr2+ to live cells was greater in the brewing than in the laboratory strain examined. However, uptake levels were greater in denatured (dried and ground) S. cerevisiae, and the relative affinities of Sr2+ for the two strains were reversed. Results for the brewing S. cerevisiae strain were similar whether the organism was obtained fresh from brewery waste or after culturing under the same conditions as for the laboratory strain. Reciprocal Langmuir plots of uptake data for live biomass were not linear, whereas those for denatured biomass were. The more complex Sr2+ binding mechanism inferred for live S. cerevisiae was underlined by cation displacement experiments. Sr2+ adsorption to live cells resulted in release of Mg2+, Ca2+, and H+, suggesting a combination of ionic and covalent bonding of Sr2+. In contrast, Mg2+ was the predominant exchangeable cation on denatured biomass, indicating primarily electrostatic attraction of Sr2+. Incubation of live S. cerevisiae in the presence of glucose resulted in a stimulation of Sr2+ uptake. Cell fractionation revealed that this increased Sr2+ uptake was mostly due to sequestration of Sr2+ in the vacuole, although a small increase in cytoplasmic Sr2+ was also evident. No stimulation or inhibition of active H+ efflux resulted from metabolism-dependent Sr2+ accumulation. However, a decline in cytoplasmic, and particularly vacuolar, Mg2+, in comparison with that of cells incubated with Sr2+ in the absence of glucose, was apparent. This was most marked for the laboratory S. cerevisiae strain, which contained higher Mg2+ levels than the brewing strain.     

Mechanisms of strontium uptake by laboratory and brewing strains of Saccharomyces cerevisiae.

Laboratory and brewing strains of Saccharomyces cerevisiae were compared for metabolism-independent and -dependent Sr2+ uptake. Cell surface adsorption of Sr2+ to live cells was greater in the brewing than in the laboratory strain examined. However, uptake levels were greater in denatured (dried and ground) S. cerevisiae, and the relative affinities of Sr2+ for the two strains were reversed. Results for the brewing S. cerevisiae strain were similar whether the organism was obtained fresh from brewery waste or after culturing under the same conditions as for the laboratory strain. Reciprocal Langmuir plots of uptake data for live biomass were not linear, whereas those for denatured biomass were. The more complex Sr2+ binding mechanism inferred for live S. cerevisiae was underlined by cation displacement experiments. Sr2+ adsorption to live cells resulted in release of Mg2+, Ca2+, and H+, suggesting a combination of ionic and covalent bonding of Sr2+. In contrast, Mg2+ was the predominant exchangeable cation on denatured biomass, indicating primarily electrostatic attraction of Sr2+. Incubation of live S. cerevisiae in the presence of glucose resulted in a stimulation of Sr2+ uptake. Cell fractionation revealed that this increased Sr2+ uptake was mostly due to sequestration of Sr2+ in the vacuole, although a small increase in cytoplasmic Sr2+ was also evident. No stimulation or inhibition of active H+ efflux resulted from metabolism-dependent Sr2+ accumulation. However, a decline in cytoplasmic, and particularly vacuolar, Mg2+, in comparison with that of cells incubated with Sr2+ in the absence of glucose, was apparent. This was most marked for the laboratory S. cerevisiae strain, which contained higher Mg2+ levels than the brewing strain.     

酵母菌对重金属离子吸附的研究

以６属３３株酵母菌的活菌或死菌对重金属离子Ｃｕ２＋、Ｃｄ２＋和Ｎｉ２＋进行了吸附能力的初步研究。结果显示∶吸附时间、吸附温度、溶液的ｐＨ、共存离子和菌体的生理状态对吸附作用都有明显的影响。在优化组合后发现一株假丝酵母菌对三种重金属离子的吸附比为Ｃｄ２＋＞Ｃｕ２＋＞Ｎｉ２＋,每克活菌体吸附量分别为１７．２３ｍｇ＞１０．５７ｍｇ＞３．２ｍｇ。干菌体对三种重金属的吸附量较明显的低于活菌体的吸附量。     

Effect of ethidium bromide and DEAE-dextran on divalent cation accumulation in yeast. Evidence for an ion-selective extrusion pump for divalent cations

The larger accumulation of Mn2+ than of Sr2+ in Saccharomyces cerevisiae is ascribed to the operation of a specific extrusion pump, presumably a Ca2+ pump, which has a higher affinity for Sr2+ than for Mn2+. The differences in accumulation levels of Mn2+ and Sr2+ attained after prolonged incubation are completely abolished in cells of which the plasmamembrane has been permeabilized with the polybase DEAE-dextran under isotonic conditions. In the permeabilized cells Sr2+ and Mn2+ accumulation levels are attained as for Mn2+ in intact cells. It is suggested that the accumulation of divalent cations into the permeabilized cells mainly represents their accumulation into the vacuoles. Also the cationic dye ethidium abolishes the differences in Mn2+ and Sr2+ accumulation. The dye increases the accumulation of Sr2+ but decreases that of Mn2+ somewhat. It cannot be distinguished yet whether its action is due to an impairment of the efflux pump or to an increase in the permeability of the plasmamembrane facilitating the divalent cations to be accumulated into the vacuoles. Ethidium does not affect the initial rates of divalent cation uptake into the vacuoles, but it effectively reduces the ultimate accumulation of the divalent cations in the DEAE-dextran permeabilized cells, possibly by competing with the divalent cations for intravacuolar binding sites. Similar results are obtained for the accumulation of Ca2+. It is concluded that the efflux pump enables the yeast cell to regulate accumulation levels of the various divalent cations to different extents.     

Process of Pb2 accumulation in Saccharomyces cerevisiae

Most of the Pb2+ taken up by Saccharomyces cerevisae was deposited in the inner part of the cells after 2 h. In the Pb2 accumulation experiments, the time to reach an equilibrium state was significantly shortened from 96 h to 24 h as the cell dry weight increased from 0.56 g/l to 5.18 g/l. The penetration time of Pb2+ to reach on the interacellular region (2 h) was quite different from that on the extracellular region (3 min). In the case of S. cerevisiae, the first step which a Pb2+ binds to cell wall within 3[f]5 min is metabolism-independent and the second step within 24 h is metabolism-dependent followed by the third step which is metabolism-dependent or -independent after 24 h.     

Rapid assessment of the physiological status of the polychlorinated biphenyl degrader Comamonas testosteroni TK102 by flow cytometry

The viability of the polychlorinated biphenyl-degrading bacterium Comamonas testosteroni TK102 was assessed by flow cytometry (FCM) with the fluorogenic ester Calcein-AM (CAM) and the nucleic acid dye propidium iodide (PI). CAM stained live cells, whereas PI stained dead cells. When double staining with CAM and PI was performed, three physiological states, i.e., live (calcein positive, PI negative), dead (calcein negative, PI positive), and permeabilized (calcein positive, PI positive), were detected. To evaluate the reliability of this double-staining method, suspensions of live and dead cells were mixed in various proportions and analyzed by FCM. The proportion of dead cells measured by FCM directly correlated with the proportion of dead cells in the sample (y = 0.9872 x + 0.18; R(2) = 0.9971). In addition, the proportion of live cells measured by FCM inversely correlated with the proportion of dead cells in the sample (y = -0.9776 x + 98.36; R(2) = 0.9962). The proportion of permeabilized cells was consistently less than 2%. These results indicate that FCM in combination with CAM and PI staining is rapid (相似文献   

Aboveground dry-matter allocation in ungrazed and grazed stands of Indigofera spinosa in the arid zone of Turkana, Kenya

The dwarf shrub Indigofera spinosa , indigenous to arid and semi-arid rangelands of northeastern Africa, is an important food source for livestock. Proper management of the shrub requires improved understanding of the effects of grazing and climatic variability on aboveground dry-matter allocation. Between 1986 and 1990, we compared the temporal variability of aboveground dry-matter allocation to different plant biomass compartments. We also compared dry-matter transfers between components; total live biomass to litter, standing dead to litter and live biomass to standing dead between continuously grazed and an ungrazed treatments. Partitioning of combined total dry-matter production among different structural organs (called allocation ratio) is influenced by phenological changes, episodic rainfall and her-bivory. Dry-matter production in the grazed treatment responded more markedly to episodic rainfall events more than in the ungrazed treatment. Exclusion of grazers failed to improve the relative growth rate (RGR) of shrub biomass, while grazing improved it. RGR declined in the ungrazed treatment following the accumulation of standing dead dry-matter, while m the grazed treatment it declined following the shedding of leaves. The shrub allocated more to total live biomass than to standing dead. Greater reduction of total live allocation ratio in the grazed than in the ungrazed treatment occurred during a dry year. The ungrazed treatment had higher standing dead allocation ratio than did the grazed treatment. Plants transferred more dry-matter from total live biomass compartment to litter, than from standing dead to or from total live biomass to standing dead independent of treatment. The rates of transfer were higher in the ungrazed than in the grazed treatment. The results suggest that I spinosa has evolved to respond to climatic variability and grazmgbyallocating dry allocating dry-matter differently between various compartments.     

外源铅、铜胁迫对不同基因型谷子幼苗生理生态特性的影响

采用盆栽土培法,研究了4种基因型谷子幼苗对Pb2+、Cu2+胁迫的生长响应、DNA损伤及吸收积累、迁移特性。结果表明,D2-8、安06、黄米和朝谷幼苗对Pb2+、Cu2+的平均耐性指数分别为0.87、0.81、0.78、0.71和0.96、0.97、0.79、0.74。在400 mg/kg Pb2+、Cu2+浓度下,4种谷子幼苗叶绿素a、b总量分别为对照的33.3%、52.6%、37.5%、49.4%和113.5%、72.3%、51.9%、75.6%,而叶绿素a/b值均高于对照。Pb2+胁迫下4种谷子幼苗中可溶性蛋白质和DNA含量随浓度升高逐渐下降,Cu2+处理组则表现为低浓度(≤100 mg/kg)的促进和高浓度(≥200 mg/kg)的抑制效应。4种谷子幼苗的DNA增色效应值在Pb2+、Cu2+胁迫下均表现为先上升后下降的趋势,其中Pb2+对朝谷和D2-8的增色效应影响较大,而Cu2+对朝谷和安06的影响最为明显。D2-8和朝谷对Pb2+、Cu2+的吸收富集能力高于安06和黄米,D2-8和安06对Pb2+、Cu2+的转运能力大于朝谷和黄米。总体来看,Pb2+对谷子幼苗的生理生态影响和遗传毒害效应大于Cu2+,4种基因型谷子对Pb2+胁迫的耐性顺序为安06>D2-8>黄米>朝谷,对Cu2+的耐性顺序为D2-8>安06>朝谷>黄米。     

Automated image analysis of live/dead staining of the fungus Aureobasidium pullulans on microscope slides and leaf surfaces

An image analysis program and protocol for the identification and enumeration of live versus dead cells of the yeast-like fungus Aureobasidium pullulans was developed for both populations on microscope slides and leaf surfaces. Live cells took up CellTracker Blue, while nonviable cells stained with DEAD Red. Image analysis macro programs running under Optimas software were used to acquire images and to differentiate and enumerate viable from nonviable cells. The software was capable of discriminating green as a third parameter for identification and quantification of green fluorescent protein-expressing cells in a wild-type population.     

酿酒酵母吸附重金属离子的研究进展

重金属污染成为当今最重要的环境问题之一。生物吸附法是处理大体积低浓度重金属废水的一种理想方法,近年来有关的研究报道不断增多,但尚未实现工业化应用。酿酒酵母(Saccharomyces cerevisiae)不仅是具有实用潜力的生物吸附剂,也是研究重金属生物吸附机理的良好材料。结合自己的研究成果,总结了酿酒酵母作为生物吸附材料的优点、研究中的表现形式和吸附性能,重点讨论了酿酒酵母生物吸附机理,介绍了等温吸附平衡模型和动力学模型在酵母生物吸附中的应用情况。最后提出生物吸附进一步的研究方向。     

Compact Cell Settlers for Perfusion Cultures of Microbial (and Mammalian) Cells

As microbial secretory expression systems have become well developed for microbial yeast cells, such as Saccharomyces cerevisiae and Pichia pastoris, it is advantageous to develop high cell density continuous perfusion cultures of microbial yeast cells to retain the live and productive yeast cells inside the perfusion bioreactor while removing the dead cells and cell debris along with the secreted product protein in the harvest stream. While the previously demonstrated inclined or lamellar settlers can be used for such perfusion bioreactors for microbial cells, the size and footprint requirements of such inefficiently scaled up devices can be quite large in comparison to the bioreactor size. Faced with this constraint, we have now developed novel, patent‐pending compact cell settlers that can be used more efficiently with microbial perfusion bioreactors to achieve high cell densities and bioreactor productivities. Reproducible results from numerous month‐long perfusion culture experiments using these devices attached to the 5 L perfusion bioreactor demonstrate very high cell densities due to substantial sedimentation of the larger live yeast cells which are returned to the bioreactor, while the harvest stream from the top of these cell settlers is a significantly clarified liquid, containing less than 30% and more typically less than 10% of the bioreactor cell concentration. Size of cells in the harvest is smaller than that of the cells in the bioreactor. Accumulated protein collected from the harvest and rate of protein accumulation is significantly (> 6x) higher than the protein produced in repeated fed‐batch cultures over the same culture duration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:913–922, 2017     

Uptake and accumulation of Mn2+ and Sr2+ in Saccharomyces cerevisiae

Initial uptake of Mn2+ and Sr2+ in the yeast Saccharomyces cerevisiae was studied in order to investigate the selectivity of the divalent cation uptake system and the possible involvement of the plasma-membrane ATPase in this uptake. The initial uptake rates of the two ions were not significantly different. This ruled out a direct role of the plasma-membrane ATPase, since this ATPase is specific for Mn2+ compared to Sr2+. After 1 h uptake, Mn2+ had accumulated 10-times more than Sr2+. Influx of Mn2+ and Sr2+ remained unchanged during that time, however. The differences in accumulation level found for Mn2+ and Sr2+ could be ascribed to a greater efflux of Sr2+ as compared with Mn2+. Probably this greater efflux of Sr2+ was only apparent, since differential extraction of the yeast cells revealed that Mn2+ is more compartmentalised than Sr2+, giving rise to a lower relative cytoplasmic Mn2+ concentration.     

Distribution of live and dead cells in pellets of an actinomycete Amycolatopsis balhimycina and its correlation with balhimycin productivity

Secondary metabolites such as antibiotics are typically produced by actinomycetes as a response to growth limiting stress conditions. Several studies have shown that secondary metabolite production is correlated with changes observed in actinomycete pellet morphology. Therefore, we investigated the correlation between the production of balhimycin and the spatio-temporal distribution of live and dead cells in pellets of Amycolatopsis balhimycina in submerged cultures. To this end, we used laser scanning confocal microscopy to analyze pellets from balhimycin producing and nonproducing media containing 0.2 and 1.0 g l^?1 of potassium di-hydrogen phosphate, respectively. We observed a substantially higher fraction of live cells in pellets from cultures yielding larger amounts of balhimycin. Moreover, in media that resulted in no balhimycin production, the pellets exhibit an initial death phase which commences from the centre of the pellet and extends in the radial direction. A second growth phase was observed in these pellets, where live mycelia are seen to appear in the dead core of the pellets. This secondary growth was absent in pellets from media producing higher amounts of balhimycin. These results suggest that distribution of live and dead cells and its correlation with antibiotic production in the non-sporulating A. balhimycina differs markedly than that observed in Streptomycetes.     

Decolorization of textile dyeing wastewater by<Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

The potential use of fungal pellets for decolorization of the textile dyeing wastewater was evaluated. The live pellets of the fungus Phanerochaete chrysosporium were found to remove more than 95% of the color of this wastewater within 1 d. The dye-removal capacity was a function of time and was proportional to the agitation rate; the optimum temperature was 30 degrees C. Both live and dead pellets were further examined in a repeated-batch mode for 5 d. The decolorization performance of live pellets remained high and stable for 5 d and they showed twice to thrice higher decolorization capacity than dead pellets.     

Stimulation of strontium accumulation in linoleate-enriched Saccharomyces cerevisiae is a result of reduced Sr2+ efflux

The influence of modified plasma membrane fatty acid composition on cellular strontium accumulation in Saccharomyces cerevisiae was investigated. Growth of S. cerevisiae in the presence of 1 mM linoleate (18:2) (which results in 18:2 incorporation to approximately 70% of total cellular and plasma membrane fatty acids, with no effect on growth rate) yielded cells that accumulated Sr2+ intracellularly at approximately twice the rate of S. cerevisiae grown without a fatty acid supplement. This effect was evident over a wide range of external Sr2+ concentrations (25 microM to 5 mM) and increased with the extent of cellular 18:2 incorporation. Stimulation of Sr2+ accumulation was not evident following enrichment of S. cerevisiae with either palmitoleate (16:1), linolenate (18:3) (n-3 and n-6 isomers), or eicosadienoate (20:2) (n-6 and n-9 isomers). Competition experiments revealed that Ca2+- and Mg2+-induced inhibition of Sr2+ accumulation did not differ between unsupplemented and 18:2-supplemented cells. Treatment with trifluoperazine (TFP) (which can act as a calmodulin antagonist and Ca2+-ATPase inhibitor), at a low concentration that precluded nonspecific K+ efflux, increased intracellular Sr2+ accumulation by approximately 3.6- and 1.4-fold in unsupplemented and 18:2-supplemented cells, respectively. Thus, TFP abolished the enhanced Sr2+ accumulation ability of 18:2-supplemented cells. Moreover, the rate of Sr2+ release from Sr2+-loaded fatty acid-unsupplemented cells was found to be at least twice as great as that from Sr2+-loaded 18:2-enriched cells. The influence of enrichment with other fatty acids on Sr2+ efflux was variable. The results reveal an enhanced Sr2+ accumulation ability of S. cerevisiae following 18:2-enrichment, which is attributed to diminished Sr2+ efflux activity in these cells.     

Calcium stores in electropermeabilized HL-60 cells before and after differentiation

Non-induced HL-60 cells (N-IND) and HL-60 cells induced to differentiate with 2 microM retinoic acid (IND) were electropermeabilized with electrical discharges, and the intracellular Ca2+ stores were measured in each type of cell. Both N-IND and IND cells accumulate Ca2+ in the presence of ATP after electropermeabilization. The Ca2+ is stored in at least two different compartments; accumulation in one of the compartments is inhibited by oligomycin and CCCP, and it is not releasable by Ins(1,4,5)P3. The maximal accumulation of Ca2+ by the Ins(1,4,5)P3 sensitive pool is about 0.3 nmol/10(6) cells and 0.9 nmol/10(6) cells for the N-IND and for the IND cells, respectively, and the half-maximal value occurs at a free Ca2+ concentration of 0.23 microM and 0.63 microM, respectively. The oligomycin + CCCP sensitive pool hardly accumulates any Ca2+ at this level of free Ca2+, but at higher free [Ca2+] (greater than microM) its maximal capacity is 80-100-fold higher than the Ins(1,4,5)P3-sensitive pool (about 17-18 nmol/10(6) cells). It is concluded that at physiological free Ca2+ concentrations, the non-mitochondrial Ca2+ pool is regulating the intracellular free Ca2+ in N-IND and IND HL-60 cells, and that this Ca2+ pool can be mobilized by Ins(1,4,5)P3. Furthermore, the capacity of this pool increases about 3-fold when the cells are induced to differentiate with retinoic acid.     

Membrane effects of phenothiazines in yeasts. I. Stimulation of calcium and potassium fluxes

Application of trifluoperazine (10-50 microM) to suspensions of the yeast Saccharomyces cerevisiae induces the following effects. (1) A marked increase in the initial rate of 45Ca2+ influx into the cells, accompanied by an increase in the cellular content of calcium. This stimulation in 45Ca2+ influx (10-20-fold) is observed only in the presence of a metabolic substrate and is completely inhibited by LaCl3. The dose-response curves of the cellular accumulation of 45Ca2+ are of a bell shape, indicating a biphasic response. The concentration of the drug yielding maximal accumulation depends on the density of the cells in the suspensions. The results indicate that the stimulation of 45Ca2+ influx is mediated by an energy-dependent carrier-mediated process and not by the increase in the passive membrane permeability to Ca2+. (2) Efflux of K+ from the cells is induced. Removal of metabolic substrate abolishes the effect at concentrations of up to 35 microM and reduces it at higher concentrations. Addition of high concentrations of cations (K+, Na+, Mg2+) to the medium abolishes the stimulation of both K+ efflux and Ca2+ influx. Chloropromazine, thioridazine and chlorprothixene display similar effects, but at higher concentrations. The results are discussed in terms of two possible alternative mechanisms; (1) calmodulin-independent effects of trifluoperazine on cell membranes, or (2) inhibition of some calmodulin-dependent processes by low concentrations of trifluoperazine.     

Enhanced Calcite Dissolution in the Presence of the Aerobic Methanotroph Methylosinus trichosporium

Microbial aerobic methane oxidation (MOx) is intrinsically coupled to the production of carbon dioxide, favoring carbonate dissolution. Recently, microbial organic polymers were shown to be able to induce carbonate dissolution. To discriminate between different mechanisms causing calcite dissolution, experiments were conducted in the presence of solid calcite with (1) actively growing cells (2) starving cells, and (3) dead cells of the methanotrophic bacterium Methylosinus trichosporium under brackish conditions (salinity 10) near calcite saturation (saturation state (Ω) 1.76 to 2.22). Total alkalinity and the amount of dissolved calcium markedly increased in all experiments containing M. trichosporium cells. After initial system equilibration, similar calcite dissolution rates, ranging between 20.16 (dead cells) and 25.68 μmol L^?1 d^?1 (actively growing cells), were observed. Although concentrations of transparent exopolymer particles declined with time in the presence of actively growing and starving cells, they increased in experiments with dead cells. Scanning electron microscopy images of calcite crystals revealed visible surface corrosion after exposure to live and dead M. trichosporium cells. The results of this study indicate a strong potential for microbial MOx to affect calcite stability negatively, facilitating calcite dissolution. In addition to CO₂ production by methanotrophically active cells, we suggest that the release of acidic or Ca²⁺-chelating organic carbon compounds from dead cells could also enhance calcite dissolution.     

The effect of EDTA and EDDS on lead uptake and localization in hydroponically grown Pisum sativum L.

Pisum sativum plants were treated for 3 days with an aqueous solution of 100 μM Pb(NO₃)₂ or with a mixture of lead nitrate and ethylenediaminetetraacetic acid (EDTA) or [S,S]-ethylenediaminedisuccinic acid (EDDS) at equimolar concentrations. Lead decline from the incubation media and its accumulation and localization at the morphological and ultrastructural levels as well as plant growth parameters (root growth, root and shoot dry weight) were estimated after 1 and 3 days of treatment. The tested chelators, especially EDTA, significantly diminished Pb uptake by plants as compared to the lead nitrate-treated material. Simultaneously, EDTA significantly enhanced Pb translocation from roots to shoots. In the presence of both chelates, plant growth parameters remained considerably higher than in the case of uncomplexed Pb. Considerable differences between the tested chelators were visible in Pb localization both at the morphological and ultrastructural level. In Pb+EDTA-treated roots, lead was mainly located in the apical parts, while in Pb+EDDS-exposed material Pb was evenly distributed along the whole root length. Transmission electron microscopy and EDS analysis revealed that in meristematic cells of the roots incubated in Pb+EDTA, large electron-dense lead deposits were located in vacuoles and small granules were rarely noticed in cell walls or cytoplasm, while after Pb+EDDS treatment metal deposits were restricted to the border between plasmalemma and cell wall. Such results imply different ways of transport of those complexed Pb forms.