A subcellular level study of copper speciation reveals the synergistic mechanism of microbial cells and EPS involved in copper binding in bacterial biofilms.

The synergistic cooperation of microbial cells and their extracellular polymeric substances (EPS) in biofilms is critical for the biofilm's resistance to heavy metals and the migration and transformation of heavy metals. However, the effects of different components of biofilms have not been fully understood. In this study, the spatial distribution and speciation of copper in the colloidal EPS, capsular EPS, cell walls and membranes, and intracellular fraction of unsaturated Pseudomonas putida (P. putida) CZ1 biofilms were fully determined at the subcellular level. It was found that 60-67% of copper was located in the extracellular fraction of biofilms, with 44.7-42.3% in the capsular EPS. In addition, there was 15.5-20.1% and 17.2-21.2% of copper found in the cell walls and membranes or the intracellular fraction, respectively. Moreover, an X-ray absorption fine structure spectra analysis revealed that copper was primarily bound by carboxyl-, phosphate-, and hydrosulfide-like ligands within the extracellular polymeric matrix, cell walls and membranes, and intracellular fraction, respectively. In addition, macromolecule quantification, fourier-transform infrared spectroscopy spectra and sulfur K-edge x-ray absorption near edge structure analysis further showed the carboxyl-rich acidic polysaccharides in EPS, phospholipids in cell walls and cell membranes, and thiol-rich intracellular proteins were involved in binding of copper in the different components of biofilm. The full understanding of the distribution and chemical species of heavy metals in biofilms not only promotes a deep understanding of the interaction mechanisms between biofilms and heavy metals, but also contributes to the development of effective biofilm-based heavy metal pollution remediation technologies.

Interaction between copper and extracellular nucleic acids in the EPS of unsaturated Pseudomonas putida CZ1 biofilm.

The role of extracellular DNA (eDNA) in biofilm in heavy metal complexation has been little reported. In this study, the interaction between the extracellular fraction of unsaturated biofilms and Cu2+ was studied using random amplified polymorphic DNA (RAPD) and synchrotron-based X-ray absorption spectroscopy (XAS) analyses. Under Cu2+ stress, the amount of eDNA was about 10-fold higher than the treatment without Cu2+ stress, which was substantially more than the amount of intracellular DNA (iDNA) present in the biofilm. The eDNA content increased significantly under Cu2+ stress and higher eDNA contents were found in colloidal extracellular polymeric substances (EPS) than in capsular EPS in Luria-Bertani medium. It was found that the composition of eDNA was distinctly changed under conditions of Cu2+ stress compared with the treatments without Cu2+ treatments, with specific eDNA bands appearing under Cu2+ treatments as revealed by RAPD analyses. X-ray absorption fine structure (XAFS) analysis assessing the molecular speciation of copper showed that copper in the secreted eDNA mainly existed as species resembling Cu3(PO4)2, followed by Cu-citrate species. This study investigated the interaction between copper and eDNA in unsaturated Pseudomonas putida CZ1 biofilms. Potential function of eDNA in biofilms under Cu2+ stress was found.

Spatial Pattern of Copper Phosphate Precipitation Involves in Copper Accumulation and Resistance of Unsaturated Pseudomonas putida CZ1 Biofilm.

Bacterial biofilms are spatially structured communities that contain bacterial cells with a wide range of physiological states. The spatial distribution and speciation of copper in unsaturated Pseudomonas putida CZ1 biofilms that accumulated 147.0 mg copper per g dry weight were determined by transmission electron microscopy coupled with energy dispersive X-ray analysis, and micro-X-ray fluorescence microscopy coupled with micro-X-ray absorption near edge structure (micro-XANES) analysis. It was found that copper was mainly precipitated in a 75 µm thick layer as copper phosphate in the middle of the biofilm, while there were two living cell layers in the air-biofilm and biofilm-medium interfaces, respectively, distinguished from the copper precipitation layer by two interfaces. The X-ray absorption fine structure analysis of biofilm revealed that species resembling Cu3(PO4)2 predominated in biofilm, followed by Cu-Citrate- and Cu-Glutathione-like species. Further analysis by micro-XANES revealed that 94.4% of copper were Cu3(PO4)2-like species in the layer next to the air interface, whereas the copper species of the layer next to the medium interface were composed by 75.4% Cu3(PO4)2, 10.9% Cu-Citrate-like species, and 11.2% Cu-Glutathione-like species. Thereby, it was suggested that copper was initially acquired by cells in the biofilm-air interface as a citrate complex, and then transported out and bound by out membranes of cells, released from the copper-bound membranes, and finally precipitated with phosphate in the extracellular matrix of the biofilm. These results revealed a clear spatial pattern of copper precipitation in unsaturated biofilm, which was responsible for the high copper tolerance and accumulation of the biofilm.

Effect of heavy-metal-resistant bacteria on enhanced metal uptake and translocation of the Cu-tolerant plant, Elsholtzia splendens.

A hydroponics trial was employed to study the effects of Pseudomonas putida CZ1 (CZ1), a heavy-metal-resistant bacterial strain isolated from the rhizosphere of Elsholtzia splendens (E. splendens), on the uptake and translocation of copper (Cu) in E. splendens. Significant promotion of plant growth coupled with the obvious plant-growth-promoting (PGP) characters of the bacteria suggested that CZ1 would be a plant-growth-promoting rhizobacterium (PGPR) to E. splendens under Cu stress condition. The results of inductively coupled plasma optical emission spectrometry (ICP-OES) showed that CZ1 increased the concentration of Cu in the shoots (up to 211.6% compared to non-inoculation treatment) and translocation factor (TF) (from 0.56 to 1.83%) of those exposed to Cu. The distribution of Cu in root cross section measured by synchrotron-based X-ray fluorescence microscopy (SRXRF) indicated that CZ1 promoted the transport of Cu from cortex to xylem in roots, which contributed to the accumulation of Cu in shoots. Furthermore, CZ1 improved the uptake of nutrient elements by plants to oppose to the toxicity of Cu. In summary, P. putida CZ1 acted as a PGPR in resistance to Cu and promoted the accumulation and translocation of Cu from root to shoot by element redistribution in plant root; hence, CZ1 is a promising assistance to phytoremediation.

Responses of unsaturated Pseudomonas putida CZ1 biofilms to environmental stresses in relation to the EPS composition and surface morphology.

The extracellular polymeric substance (EPS) and surface properties of unsaturated biofilms of a heavy metal-resistant rhizobacterium Pseudomonas putida CZ1, in response to aging, pH, temperature and osmotic stress, were studied by quantitative analysis of EPS and atomic force microscope. It was found that EPS production increased approximately linearly with culture time, cells in the air-biofilm interface enhanced EPS production and decreased cell volume to cope with nutrient depletion during aging. Low pH, high temperature and certain osmotic stress (120 mM NaCl) distinctly stimulated EPS production, and the main component enhanced was extracellular protein. In addition to the enhancement of EPS production in response to high osmotic (328 mM NaCl) stress, cells in the biofilm adhere tightly together to maintain a particular microenvironment. These results indicated the variation of EPS composition and the cooperation of cells in the biofilms is important for the survival of Pseudomonas putida CZ1 from environmental stresses in the unsaturated environments such as rhizosphere.

Morphological alterations of Vero cell exposed to coplanar PCB 126 and noncoplanar PCB 153.

Polychlorinated biphenyls (PCBs) are widespread, persistent environmental contaminants that display a complex spectrum of toxicological properties. Exposure to PCBs has been associated with morphological anomalies in cell cultures. However, most mechanistic studies of PCBs' toxic activity have been focused on coplanar congeners. It is of importance to determine whether PCB treatment would influence cell configuration and whether these changes would depend on the structural characteristics of PCBs. In this study, we investigated cell morphological alteration in Vero cell cultures after exposure to coplanar PCB 126 and noncoplanar PCB 153. The survival of Vero cells was measured through the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test. Cytotoxicity results suggested that PCB congeners had a toxic, antiproliferative effect on Vero cells. Morphological studies described structural modifications and provided evidence that apoptosis might be the main cell death pathway in PCB 153-treated cells. The comparison between PCB 126 and PCB 153 indicated that the cell death mechanisms involved in coplanar or noncoplanar PCB congener exposure were different in Vero cells.

Assessment of phenanthrene bioavailability in aged and unaged soils by mild extraction.

It has become apparent that the threat of an organic pollutant in soil is directly related to its bioavailable fraction and that the use of total contaminant concentrations as a measure of potential contaminant exposure to plants or soil organisms is inappropriate. In light of this, non-exhaustive extraction techniques are being investigated to assess their appropriateness in determining bioavailability. To find a suitable and rapid extraction method to predict phenanthrene bioavailability, multiple extraction techniques (i.e., mild hydroxypropyl-ß-cyclodextrin (HPCD) and organic solvents extraction) were investigated in soil spiked to a range of phenanthrene levels (i.e., 1.12, 8.52, 73, 136, and 335 µg g( - 1) dry soil). The bioaccumulation of phenanthrene in earthworm (Eisenia fetida) was used as the reference system for bioavailability. Correlation results for phenanthrene suggested that mild HPCD extraction was a better method to predict bioavailability of phenanthrene in soil compared with organic solvents extraction. Aged (i.e., 150 days) and fresh (i.e., 0 day) soil samples were used to evaluate the extraction efficiency and the effect of soil contact time on the availability of phenanthrene. The percentage of phenanthrene accumulated by earthworms and percent recoveries by mild extractants changed significantly with aging time. Thus, aging significantly reduced the earthworm uptake and chemical extractability of phenanthrene. In general, among organic extractants, methanol showed recoveries comparable to those of mild HPCD for both aged and unaged soil matrices. Hence, this extractant can be suitable after HPCD to evaluate risk of contaminated soils.

Adsorption of copper and zinc by biochars produced from pyrolysis of hardwood and corn straw in aqueous solution.

Biochars produced by pyrolysis of hardwood at 450 °C (HW450) and corn straw at 600 °C (CS600) were characterized and investigated as adsorbents for the removal of Cu(II) and Zn(II) from aqueous solution. The adsorption data were well described by a Langmuir isotherm, with maximum Cu(II) and Zn(II) adsorption capacities of 12.52 and 11.0 mg/g for CS600, 6.79 and 4.54 mg/g for HW450, respectively. Thermodynamic analysis suggested that the adsorption was an endothermic process and did not occur spontaneously. Although Cu(II) adsorption was only marginally affected by Zn(II), Cu(II) competed with Zn(II) for binding sites at Cu(II) and Zn(II) concentrations ≥ 1.0mM. Results from this study indicated that plant-residue or agricultural waste derived biochar can act as effective surface sorbent, but their ability to treat mixed waste streams needs to be carefully evaluated on an individual basis.

Sorption and distribution of copper in unsaturated Pseudomonas putida CZ1 biofilms as determined by X-ray fluorescence microscopy.

The spatial and temporal distribution of metals in unsaturated Pseudomonas putida CZ1 biofilms was determined using synchrotron-based X-ray fluorescence microscopy (XRF). It was found that Fe, Mn, and Ca were mainly distributed near the air-biofilm interface of a biofilm grown on 40 mM citrate, while there were two Fe-, Mn-, and Ca-rich layers within a biofilm grown on 10 mM citrate. The sorption of copper by biofilm grown in medium containing 10 mM citrate was rapid, with copper being found throughout the biofilm after only 1 h of exposure. Copper initially colocalized with Fe and Mn element layers in the biofilm and then precipitated in a 40-µm-thick layer near the air-biofilm interface when exposed for 12 h. Cu K-edge X-ray absorption near edge structure (XANES) analysis revealed that Cu was primarily bound with citrate within the biofilm, and the precipitate formed in the biofilm exposed to copper for 12 h was most similar to copper phosphate. LIVE/DEAD staining revealed that cells at the biofilm-membrane interface were mostly alive even when the copper concentration reached 80.5 mg copper g(-1) biomass. This suggests that the biofilm matrix provided significant protection for cells in this area. These results significantly improve our understanding of metal acquisition, transportation, and immobilization in unsaturated biofilm systems.

Enhancement of copper availability and microbial community changes in rice rhizospheres affected by sulfur.

The role of sulfur on the availability of Cu and the bacterial community in rice rhizospheres was investigated by pot experiments. With sulfur addition, pH in rhizosphere soil decreased and Mg(NO3)2 extractable Cu increased significantly. The bacterial community composition also changed with sulfur addition. Some specific clones having high similarity to Thiobacillus, which indicated that sulfur oxidation in the rice rhizosphere could increase the availability of Cu. These results suggested that sulfur source which could provide substrate to sulfur oxidizing bacteria and enhance the availability of Cu was not a suitable sulfur fertilizer for Cu polluted soil.

Assessment of pyrene bioavailability in soil by mild hydroxypropyl-ß-cyclodextrin extraction.

Bioavailability of organic pollutants in soil is currently a much-debated issue in risk assessment of contaminated sites. Ecorisk of an organic pollutant in soil is strongly influenced by the properties of the soil and its contamination history. To evaluate the effect of aging on the availability of pyrene, earthworm (Eisenia fetida) accumulation and chemical extraction by exhaustive and nonexhaustive techniques in soil spiked with a range of pyrene levels (1.07, 9.72, 88.4, 152, and 429 µg g⁻¹ dry soil) were measured in this study using both unaged (i.e., 0 days) and aged (i.e., 69, 150, and 222 days) soil samples. The results showed that the amount of pyrene accumulated by earthworms did not change greatly with aging time under different high-dose contamination levels, but changed significantly at lower concentrations. Moreover, aging (after 222 days) significantly decreased biological and chemical availability of pyrene. Furthermore, the relationship between earthworm bioaccumulation, hydroxypropyl-ß-cyclodextrin (HPCD), and organic solvent extraction was investigated in order to find a suitable and rapid method to predict pyrene bioavailability. Results showed that, at different levels of pyrene, the mean values of earthworm uptake and HPCD extractability were 10-40% and 10-65%, respectively. Correlation (r² = 0.985) and extraction results for pyrene suggested that mild HPCD extraction was a better method to predict bioavailability of pyrene in soil compared with organic solvent extraction.

Copper uptake and its effect on metal distribution in root growth zones of Commelina communis revealed by SRXRF.

To explore the copper uptake mechanisms by the Cu-tolerant plant Commelina communis, the contents of Cu and other metals (including Fe, Zn, and Mn) in roots were detected using atomic absorption spectrometer under transporter inhibitors, partial element deficiency, or Cu excess treatments, while distribution characters of Cu and other metals in root growth zones were investigated by synchrotron radiation X-ray fluorescence spectroscopy (SRXRF). Cu uptake was inhibited by the uncoupler DNP and P-type ATPase inhibitor Na(3)VO(4), not by the Ca(2+) ion channel inhibitor LaCl(3), suggesting that Cu could probably be assimilated actively by root and be related with P-type ATPase, but not through Ca(2+) ion channel. Fe or Zn deficiency could enhance Cu uptake, while 100 µM Cu inhibited Fe, Zn, and Mn accumulation in roots significantly. Metal distribution under 100 µM Cu treatment was investigated by SRXRF. High level of Cu was found in the root meristem, and higher Cu concentrations were observed in the vascular cylinder than those in the endodermis, further demonstrating the initiative Cu transport in the root of C. communis. Under excess Cu stress, most Fe was located in the epidermis, and Fe concentrations in the endodermis were higher than those in the vascular cylinder, suggesting Cu and Fe competition not only in the epidermal cells but also for the intercellular and intracellular transport in roots. Zn was present in the meristem and the vascular cylinder similar to Cu. Cu and Zn showed a similar pattern. Mn behaves as Zn does, but not like Fe.

Subcellular distribution and chemical forms of cadmium in Phytolacca americana L.

Phytolacca americana L. (pokeweed) is a promising species for Cd phytoextraction with large biomass and fast growth rate. To further understand the mechanisms involved in Cd tolerance and detoxification, the present study investigated subcellular distribution and chemical forms of Cd in pokeweed. Subcellular fractionation of Cd-containing tissues indicated that both in root and leaves, the majority of the element was located in soluble fraction and cell walls. Meanwhile, Cd taken up by pokeweed existed in different chemical forms. Results showed that the greatest amount of Cd was found in the extraction of 80% ethanol in roots, followed by 1 M NaCl, d-H(2)O and 2% HAc, while in leaves and stems, most of the Cd was extracted by 1 M NaCl, and the subdominant amount of Cd was extracted by 80% ethanol. It could be suggested that Cd compartmentation with organo-ligands in vacuole or integrated with pectates and proteins in cell wall might be responsible for the adaptation of pokeweed to Cd stress.

Curli produced by Escherichia coli PHL628 provide protection from Hg(II).

The role of curli, amyloid extracellular fibers, in the tolerance of Escherichia coli PHL628 to Hg(II) was examined. Our findings indicate that by sorbing Hg(II) extracellularly, curli protect the cells. To our knowledge, this is the first time a protective role of curli against toxic metals has been demonstrated.

Lead contamination in different varieties of tea plant (Camellia sinensis L.) and factors affecting lead bioavailability.

BACKGROUND: There has been increasing concern in recent years about the concentration of lead (Pb) in tea. However, little research has been done to address questions concerning the distribution of Pb in different varieties of tea plant and the differences among tea plant varieties in their uptake and accumulation of Pb from the soil. Therefore the aim of this study was to investigate the accumulation of Pb in different tea plant varieties and the factors affecting Pb bioavailability. RESULTS: Three patterns of Pb distribution could be observed in different plant parts among the eight tea varieties surveyed, as well as a linear relationship between the Pb concentration in fine roots and the exchangeable Pb fraction in the corresponding soil. The uptake of Pb by fine roots increased significantly as the soil pH decreased. The average ratios of Pb concentration in fine roots to those in young stems and young leaves were 5.18 and 31.80 respectively. In fine roots the Pb concentration varied from 22.7 to 61.6 mg kg(-1). CONCLUSION: The results indicated that the uptake, transport and accumulation of Pb by tea plant organs were strongly governed by soil conditions and tea variety, thus providing tea producers with useful information on variety selection for the production of quality teas containing low levels of Pb.

Comparison of structure-dependent hormetic cytotoxicity induced by coplanar and non-coplanar PCB congeners.

The effect of polychlorinated biphenyls (PCBs) on Vero cell proliferation was investigated, with the attempts to assess the possible hormetic dose-response and to compare their structure-dependent toxicity. Both PCB congeners revealed low doses stimulation in our experiment. However, significant cytotoxicity was only observed in PCB 52 concentrations larger than 0.1 microg ml(-1), while there was no significant inhibition in PCB 77-treated cells at concentrations selected. Furthermore, the time-dependent cytotoxic trends were different. The comparison between PCB 52 and PCB 77 indicated that the cytotoxic mechanisms involved in coplanar or non-coplanar PCB congener exposure were different, and this difference might be associated with individual genotoxicity and the release of contact inhibition, respectively.

[Persisters and their effects on microbial biofilm tolerance: a review].

Persisters are a group of special subpopulation of bacteria, only occupying < 0.1% of the whole population but having the characteristics different from the ordinary bacteria and resistant mutants. They have complex formation mechanism, and are difficult to isolate and culture. The persisters can adapt to the adverse environment via "dormancy-growth-proliferation" to maintain their survival and cell structure stability, and play a vital role in the multi-drug and multi-metal tolerance of microbial biofilm, being of great significance in maintaining the stability of microbial community structure. This paper reviewed the research progress on the characteristics of persisters, their gene regulation mechanisms, and their effects on the multi-drug and multi-metal tolerance of microbial biofilm. The related research directions in the future were also prospected.

Enhanced phytoremediation potential of polychlorinated biphenyl contaminated soil from e-waste recycling area in the presence of randomly methylated-beta-cyclodextrins.

The crude recycling of electronic and electric waste (e-waste) is now creating soil pollution problems with organic compounds such as polychlorinated biphenyls (PCBs). The present study aimed to compare the phytoremediation potential of four plant species (rice, alfalfa, ryegrass and tall fescue) for PCBs contaminated soil from Taizhou city, one of the largest e-waste recycling centers in China. In addition, the enhanced effects of randomly methylated-beta-cyclodextrins (RAMEB) on PCBs phytoremediation potential were evaluated. Higher PCBs removal percentages of 25.6-28.5% in rhizosphere soil were observed after 120 days, compared with those of the non-rhizosphere (10.4-16.9%) and unplanted controls (7.3%). The average PCBs removal percentages of four plant species increased from 26.9% to 37.1% in the rhizosphere soil with addition of RAMEB. Meanwhile, relatively high microbial counts and dehydrogenase activity were detected in planted soils and a stimulatory effect by RAMEB addition was found. The present study indicated that all the plant candidates were feasible for phytoremediation of PCBs contaminated soil from the e-waste recycling area, and tall fescue with RAMEB amendment seemed as a promising remediation strategy. High PCBs removal percentage was due to the increased PCBs bioavailability as well as biostimulation of microbial communities after plantation and RAMEB addition.

Hormesis response of marine and freshwater luminescent bacteria to metal exposure.

The stimulatory effect of low concentrations of toxic chemicals on organismal metabolism, referred to as hormesis, has been found to be common in the widely used luminescence bioassay. This paper aims to study the hormesis phenomenon in both marine and freshwater luminescent bacteria, named Photobacterium phosphorem and Vibrio qinghaiensis. The effects of Cu (II), Zn (II), Cd (II) and Cr (VI) on luminescence of these two bacteria were studied for 0 to 75 minutes exposure by establishing dose- and time-response curves. A clear hormesis phenomenon was observed in all four testing metals at low concentrations under the condition of luminescence assays.

Enhancement of phenanthrene and pyrene degradation in rhizosphere of tall fescue (Festuca arundinacea).

A greenhouse experiment was conducted with varying concentrations of phenanthrene (11-344 mg kg(-1)) and pyrene (15-335 mg kg(-1)) spiked in the soil to evaluate the phytoremediation of PAHs contaminated soil using tall fescue (Festuca arundinacea). After 65-day of tall fescue growth, plant biomass, microbial viable counts, dehydrogenase activity, water-soluble phenolic compounds, phenanthrene and pyrene residual concentrations and removal percentages were determined. The results showed that target PAHs (phenanthrene and pyrene) did not affect plant biomass at lower concentrations but a reduced biomass (only 53.5% of shoot and 29.7% of root compared to control) was observed at higher concentrations. Higher biological activities (microbial viable counts, water-soluble phenolic compounds, dehydrogenase activity) and PAHs degradation rates were detected in planted soils than unplanted controls. After harvest, 91.7-97.8% of phenanthrene and 70.8-90.0% of pyrene had been degraded in the planted soils, which were 1.88-3.19% and 8.85-20.69% larger than those in corresponding unplanted soils. This enhanced dissipation of target PAHs in planted soils might be derived from increased biological activity in the rhizosphere. The results of the present study suggest that the presence of tall fescue roots were effective in promoting the phytoremediation of PAHs contaminated soil.