High Metal Tolerance Research Articles

Utilizing metal tolerance potential of soil fungus for efficient synthesis of gold nanoparticles with superior catalytic activity for degradation of rhodamine B

In recent years, the surging demand of nanomaterials has boosted unprecedented expansion of research for the development of high yielding and sustainable synthesis methods which can deliver nanomaterials with desired characteristics. Unlike the well-established physico-chemical methods which have various limitations, biological methods inspired by mimicking natural biomineralization processes have great potential for nanoparticle synthesis. An eco-friendly and sustainable biological method that deliver particles with well-defined shape, size and compositions can be developed by selecting a proficient organism followed by fine tuning of various process parameter. The present study revealed high metal tolerance ability of a soil fungus Cladosporium oxysporum AJP03 and its potential for extracellular synthesis of gold nanoparticles. The morphology, composition and crystallinity of nanoparticles were confirmed using standard techniques. The synthesized particles were quasi-spherical in shape with fcc packing and an average particle size of 72.32 ± 21.80 nm. A series of experiments were conducted to study the effect of different process parameters on particle size and yield. Biomass: water ratio of 1:5 and 1 mM precursor salt concentration at physiological pH (7.0) favoured the synthesis of well-defined gold nanoparticles with maximum yield. The as-synthesized nanoparticles showed excellent catalytic efficiency towards sodium borohydride mediated reduction of rhodamine B (2.5 × 10−5 M) within 7 min of reaction time under experimental conditions. Presence of proteins as capping material on the nanoparticle surface was found to be responsible for this remarkable catalytic efficiency. The present approach can be extrapolated to develop controlled and up-scalable process for mycosynthesis of nanoparticles for diverse applications.

The green alga Zygogonium ericetorum (Zygnematophyceae, Charophyta) shows high iron and aluminium tolerance: protection mechanisms and photosynthetic performance.

Streptophyte green algae, ancestors of Embryophytes, occur frequently in terrestrial habitats being exposed to high light intensities, water scarcity and potentially toxic metal cations under acidic conditions. The filamentous Zygogonium ericetorum synthesizes a purple vacuolar ferrous pigment, which is lost after aplanospore formation. However, it is unknown whether this cellular reorganization also removes excessive iron from the protoplast and how Z. ericetorum copes with high concentrations of aluminium. Here we show that aplanospore formation shifts iron into the extracellular space of the algal filament. Upon germination of aplanospores, aluminium is bound in the parental cell wall. Both processes reduce iron and aluminium in unpigmented filaments. Comparison of the photosynthetic oxygen production in response to light and temperature gradients in two different Z. ericetorum strains from an Austrian alpine and a Scottish highland habitat revealed lower values in the latter strain. In contrast, the Scottish strain showed a higher optimum quantum yield of PSII during desiccation stress followed by rehydration. Furthermore, pigmented filaments of both strains exhibited a higher light and temperature dependent oxygen production when compared to the unpigmented phenotype. Our results demonstrate a high metal tolerance of Z. ericetorum, which is crucial for surviving in acidic terrestrial habitats.

New mutualistic fungal endophytes isolated from poplar roots display high metal tolerance.

This study aimed to isolate, identify, and characterise metal-tolerant fungi colonising poplar roots at a metal-contaminated phytoremediation site. Poplar roots were colonised by arbuscular mycorrhizal, ectomycorrhizal, and endophytic fungi, and the species were determined by ITS molecular analyses. Eight different isolates were successfully isolated into pure culture. Three isolates belonging to the Helotiales (P02, P06) and the Serendipita vermifera species (P04) were highly tolerant to metals (Cd, Zn, Pb, and Cu) compared to the mycorrhizal Hebeloma isolates. The three isolates degraded complex carbohydrates, such as xylan and cellulose, indicating that they could partially degrade root cell walls and penetrate into cells. This hypothesis was confirmed by further in vitro re-synthesis experiments, which showed that the three isolates colonised root tissues of poplar plantlets whereas two of them formed microsclerotia-like structures. Taken together, these results suggest an endophytic lifestyle of these isolates. This is the first evidence of S. vermifera as a root endophyte of poplar. A new endophytic putative species belonging to the Helotiales and closely related to Leohumicola is also reported. Interestingly, and when compared to mock-inoculated plants, both P06 and P04 isolates increased the number of root tips of inoculated poplar plantlets in vitro. Moreover, the S. vermifera P04 isolate also increased the shoot biomass. The results are discussed in relation to the potential use of endophytic strains for tree-based phytoremediation of metal-contaminated sites.

An efficient approach towards the bioremediation of copper, cobalt and nickel contaminated field samples

The synthetic soil based bioremediation approach as reasonable and sustainable practice at the farming level where desired bioremediation could be established at lower cost. Metal-tolerant bacteria from different environmental field samples, (a) a municipal dump site, (b) an agricultural field and (c) sludge of electro-plating industries, were screened and characterized. Bioremediation of metal contaminants through isolated bacteria was compared under two different conditions, synthetic soil and basic minimal media containing copper, cobalt and nickel. The pollutants arising from industrial effluents are imparting a huge negative impact on agricultural land. Microbes are predominant in heavy metal-contaminated sites, which signifies as a potential opportunity for the researchers towards bioremediation. Three bacterial species showed high metal tolerance; 16S ribosomal DNA (rDNA) analysis revealed that the organisms were Proteus vulgaris strain, Stenotrophomonas sp. and Bacillus thuringiensis. Percentage removal of metals was also analysed under different concentrations and pH. The current tested methods are helpful in streamlining the natural compliance of fragile elements and its uptake into the microbial system under in vitro and in situ conditions.

Synthesis of AgNPs using Aspergillus terreus and Evaluation of Its Antimicrobial Activity

Extracellular mycosynthesis of Ag Nano crystals by using the biological organism Aspergillus terreus is one of the most environmental friendly approaches as Aspergillus terreus can easily be cultured on simple media nutrients and shows high metal tolerance. Downstream processing for recovery of extracellular Ag nanocrystals is simpler making the process commercially feasible. Silver nanoparticles fabrication was characterized using UV-visible spectrophotometer and maximum absorbtion was obtained at 450 nm. Nitrate reductase test was used to confirm the presence of reductase protein involved in Ag nanoparticles fabrication. These mycosynthesized nanoparticles were found to have broad antimicrobial spectrum against pathogens including C. albicans, C. tropicalls, C. parapsilosis, C. glabrata, C. krusei, A. flavus, E. coli, P. aeruginosa and S. aureus as determined by agar diffusion method.

Model systems to unravel the molecular mechanisms of heavy metal tolerance in the ericoid mycorrhizal symbiosis.

Ericoid mycorrhizal plants dominate in harsh environments where nutrient-poor, acidic soil conditions result in a higher availability of potentially toxic metals. Although metal-tolerant plant species and ecotypes are known in the Ericaceae, metal tolerance in these plants has been mainly attributed to their association with ericoid mycorrhizal fungi. The mechanisms underlying plant protection by the fungal symbiont are poorly understood, whereas some insights have been achieved regarding the molecular mechanisms of heavy metal tolerance in the fungal symbiont. This review will briefly introduce the general features of heavy metal tolerance in mycorrhizal fungi and will then focus on the use of "omics" approaches and heterologous expression in model organisms to reveal the molecular bases of fungal response to heavy metals. Functional complementation in Saccharomyces cerevisiae has allowed the identification of several ericoid mycorrhizal fungi genes (i.e., antioxidant enzymes, metal transporters, and DNA damage repair proteins) that may contribute to metal tolerance in a metal-tolerant ericoid Oidiodendron maius isolate. Although a powerful system, the use of the yeast complementation assay to study metal tolerance in mycorrhizal symbioses has limitations. Thus, O. maius has been developed as a model system to study heavy metal tolerance mechanisms in mycorrhizal fungi, thanks to its high metal tolerance, easy handling and in vitro mycorrhization, stable genetic transformation, genomics, transcriptomic and proteomic resources.

Cytoplasmic CopZ-Like Protein and Periplasmic Rusticyanin and AcoP Proteins as Possible Copper Resistance Determinants in Acidithiobacillus ferrooxidans ATCC 23270.

Acidophilic organisms, such as Acidithiobacillus ferrooxidans, possess high-level resistance to copper and other metals. A. ferrooxidans contains canonical copper resistance determinants present in other bacteria, such as CopA ATPases and RND efflux pumps, but these components do not entirely explain its high metal tolerance. The aim of this study was to find other possible copper resistance determinants in this bacterium. Transcriptional expression of A. ferrooxidans genes coding for a cytoplasmic CopZ-like copper-binding chaperone and the periplasmic copper-binding proteins rusticyanin and AcoP, which form part of an iron-oxidizing supercomplex, was found to increase when the microorganism was grown in the presence of copper. All of these proteins conferred more resistance to copper when expressed heterologously in a copper-sensitive Escherichia coli strain. This effect was absent when site-directed-mutation mutants of these proteins with altered copper-binding sites were used in this metal sensitivity assay. These results strongly suggest that the three copper-binding proteins analyzed here are copper resistance determinants in this extremophile and contribute to the high-level metal resistance of this industrially important biomining bacterium.

Cloning and functional analysis of MxNRAMP1 and MxNRAMP3, two genes related to high metal tolerance of Malus xiaojinensis

Metal stresses are common nutritional disorder in plants, especially in fruit trees grown in calcareous soil. The natural resistance-associated macrophage protein (NRAMP) family comprises integral membrane proteins that play an important role in regulating metal ion transport. Our research focused on an iron deficiency-resistant apple (Malus xiaojinensis) rootstock, as the study model. Based on conserved regions of NRAMP1 and NRAMP3 in other plants, we designed primers and cloned MxNRAMP1 and MxNRAMP3 genes from M. xiaojinensis, the functions of which were preliminarily characterized in yeast, a eukaryotic model organism. In this study, MxNRAMP1 is localized to the plasma membrane, whereas MxNRAMP3 is localized to the vacuolar membrane. In yeast, both proteins were able to transport Cd but not other metal ions. Upon Fe, Cd, Mn, and Zn stress treatments for M. xiaojinensis, MxNRAMP1 expression was specifically induced in roots. MxNRAMP3 was expressed in both roots and leaves, and was immediately expressed in roots at the onset of metal stress, whereas its expression was delayed in leaves. These results showed that MxNRAMP1 and MxNRAMP3 play an important role in metal absorption and transportation.

Lead accumulation and tolerance of Moso bamboo (Phyllostachys pubescens) seedlings: applications of phytoremediation.

A hydroponics experiment was aimed at identifying the lead (Pb) tolerance and phytoremediation potential of Moso bamboo (Phyllostachys pubescens) seedlings grown under different Pb treatments. Experimental results indicated that at the highest Pb concentration (400 μmol/L), the growth of bamboo seedlings was inhibited and Pb concentrations in leaves, stems, and roots reached the maximum of 148.8, 482.2, and 4282.8 mg/kg, respectively. Scanning electron microscopy revealed that the excessive Pb caused decreased stomatal opening, formation of abundant inclusions in roots, and just a few inclusions in stems. The ultrastructural analysis using transmission electron microscopy revealed that the addition of excessive Pb caused abnormally shaped chloroplasts, disappearance of endoplasmic reticulum, shrinkage of nucleus and nucleolus, and loss of thylakoid membranes. Although ultrastructural analysis revealed some internal damage, even the plants exposed to 400 µmol/L Pb survived and no visual Pb toxicity symptoms such as necrosis and chlorosis were observed in these plants. Even at the highest Pb treatment, no significant difference was observed for the dry weight of stem compared with controls. It is suggested that use of Moso bamboo as an experimental material provides a new perspective for remediation of heavy metal contaminated soil owing to its high metal tolerance and greater biomass.

Comparison of manganese tolerance and accumulation among 24 Salix clones in a hydroponic experiment: Application for phytoremediation

Willows (Salix spp.) are excellent candidates for phytoremediation owing to their large biomass, high metal tolerance and accumulation capacity. In this study, manganese tolerance and accumulation capability in 24 Salix clones were evaluated exposed to 1mM Mn by a hydroponic system for 21days. Results suggested that there were wide variations in Mn tolerance and accumulation capability among the clones. Clonal variation in biomass production ranged from growth reduction to growth stimulation. The clonal differences in Mn concentrations (μgg−1, dry weight, DW) ranged from 3183.10 to 5827.7 in leaves, from 1840.48 to 4572.17 in stems, and from 2733.33 to 10,253.88 in roots exposed to excess Mn. The total Mn contents in shoots (including leaves and stems) varied 5.8-fold among clones under Mn treatment. Five clones exhibited high Mn tolerance and accumulation capacity, and clone J333 (Salix babylonica×Salix matsudana) had a relatively high Mn tolerance index and the highest Mn content in aboveground tissues. Consequently, further evaluation of the Salix clones for Mn tolerance and phytoremediation potential is recommended in field experiments.

Phytoremediation of Pb, Zn, Fe, and Mg with 25 wetland plant species from a paper mill contaminated site in North East India.

In order to assess the potential of wetland plants to remediate metals from a paper mill effluent contaminated wetland site in Northeast India, 25 abundant plant species belonging to 15 different families, soil, and water samples from the sites were tested for Pb, Zn, Mg, and Fe by atomic absorption spectrophotometer. The results showed that metal accumulation by wetland plants differed among species and tissues. Plants thrived in high Pb, Zn, Mg, and Fe which indicated their tolerance. According to the criteria used for selecting plants for phytoremediation such as high metal tolerance, short life cycle, wide distribution, large shoot biomass and translocation factor (TF) >1; five species each were Mg and Fe accumulators, nine species were Pb accumulators and, eight species were Zn accumulators and the rest were excluders. Alternanthera sessilis was the only plant species that had TF > 1 for all the four metals. The study indicated great promise for phytoremediation, as these accumulators could be used in future for practical phytoremediation approaches and reduction of the risk from harmful metals to human health.

A biomimetic approach towards synthesis of zinc oxide nanoparticles

Using natural processes as inspiration, the present study demonstrates a positive correlation between zinc metal tolerance ability of a soil fungus and its potential for the synthesis of zinc oxide (ZnO) nanoparticles. A total of 19 fungal cultures were isolated from the rhizospheric soils of plants naturally growing at a zinc mine area in India and identified on the genus, respectively the species level. Aspergillus aeneus isolate NJP12 has been shown to have a high zinc metal tolerance ability and a potential for extracellular synthesis of ZnO nanoparticles under ambient conditions. UV-visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction analysis, transmission electron microscopy, and energy dispersive spectroscopy studies further confirmed the crystallinity, morphology, and composition of synthesized ZnO nanoparticles. The results revealed the synthesis of spherical nanoparticles coated with protein molecules which served as stabilizing agents. Investigations on the role of fungal extracellular proteins in the synthesis of nanoparticles indicated that the process is nonenzymatic but involves amino acids present in the protein chains.

Bioaccumulation and glutathione-mediated detoxification of copper and cadmium in Sphagnum squarrosum Crome Samml.

Physiological and biochemical responses, metal bioaccumulation and tolerance potential of Sphagnum squarrosum Crome Samml. to Cu and Cd were studied to determine its bioindication and bioremediation potential. Results suggest that glutathione treatment increases the metal accumulation potential and plays a definite role in heavy metal scavenging. High abundance of Sphagnum in metal-rich sites strongly suggests its high metal tolerance capabilities. This experiment demonstrates that S. squarrosum is able to accumulate and tolerate a high amount of metals and feasibility of its application as bioindicator and remediator test species of metal-contaminated environment.

Cytoplasmic membrane response to copper and nickel in Acidithiobacillus ferrooxidans

Metal tolerance has been found to vary among Acidithiobacillus ferrooxidans strains and this can impact the efficiency of biomining practices. To explain observed strain variability for differences in metal tolerance we examined the effects of Cu 2+ and Ni 2+ concentrations (1–200 mM) on cytoplasmic membrane properties of two A. ferrooxidans type strains (ATCC 23270 and 19859) and four strains isolated from AMD water around Sudbury, Ontario, Canada. Growth rate, membrane fluidity and phase, determined from the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), and fatty acid profiles indicated that three different modes of adaptation were present and could separate between strains showing moderate, or high metal tolerance from more sensitive strains. To compensate for the membrane ordering effects of the metals, significant remodelling of the membrane was used to either maintain homeoviscous adaptation in the moderately tolerant strains or to increase membrane fluidity in the sensitive strains. Shifts in the gel-to-liquid crystalline transition temperature in the moderately tolerant strains led to multiple phase transitions, increasing the potential for phase separation and compromised membrane integrity. The metal-tolerant strain however, was able to tolerate increases in membrane order without significant compensation via fatty acid composition. Our multivariate analyses show a common adaptive response which involves changes in the abundant 16:0 and 18:1 fatty acids. However, fatty acid composition and membrane properties showed no difference in response to either copper or nickel suggesting that adaptive response was non-specific and tolerance dependent. We demonstrate that strain variation can be evaluated using differences in membrane properties as intrinsic determinants of metal susceptibility.

Ectomycorrhizal community structure under willows at former ore mining sites

The goat willow ( Salix caprea) belongs to the typical pioneer vegetation at former ore mining sites in Middle Europe as a result of its high metal-tolerance. Ectomycorrhizal (EM) fungi are known to contribute essentially to the tolerance of plants against abiotic stress. We investigated the EM associations of S. caprea at three former silver-mining sites around Freiberg (Saxonia, Germany) after 33 years of revegetation. The EM colonisation ranged from 3 to 36% of the fine root tips. We found 14 different EM types and characterised them morphologically as well as anatomically. The fungal partners were identified using LSU and ITS sequences. They belonged to four families: Thelephoraceae, Cortinariaceae, Tricholomataceae and Tuberaceae. Thelephoraceae, with 53–91%, comprised the largest portion of the total EM colonisation at all test sites and sampling dates. Thelephoraceae produced mainly contact exploration types of ectomycorrhiza. Tricholoma cingulatum was the only EM fungus which was found as ectomycorrhiza and sporocarp. We suppose that the low exploration of the majority of EM associations of S. caprea, in combination with high portions of metal-chelating organic acids containing fungal partners, contributes essentially to the high tolerance of this tree species against high metal concentrations in the soil.

Differential Genetic Expression in Heap Bioleaching of Low-Grade Copper Sulphide Ore at Escondida Mine, Chile

During the last decades, microbial bioleaching of metallic sulphide ores has become a very important process in the mining industry. The study of biodiversity in those systems has revealed the occurrence of several acidophilic organisms, forming a dynamic community including Bacteria and Archaea. Despite the importance of the heap bioleaching process, the metabolic behaviour of a microbial community in bioleaching systems remains unknown. The role played by members of the community inhabiting a bioheap environment has been estimated by the phenotypic characteristics of pure cultures, such as iron oxidation, sulphur oxidation, resistance to high ion concentration and metal tolerance, among others. The global genomic expression of the community inhabiting an industrial bioheap of low-grade copper sulphide ore at Escondida mine in Chile was investigated by random arbitrary primed polymerase chain reaction (RAP-PCR). Three random primers were used and twenty differentially expressed bands were cloned and sequenced. Several sequences were related to 16S rRNA of members of the microbial community; two sequences were related to the α subunit of pyruvate dehydrogenase from Acidithiobacillus ferrooxidans. The expression levels of the pyruvate dehydrogenase gene in samples from an industrial bioleaching operation at different stages were checked by quantitative real-time PCR.

Body mass and caloric value of the ground beetle (Pterostichus oblongopunctatus) (Coleoptera, Carabidae) along a gradient of heavy metal pollution

At five sites located along a metal-pollution gradient in southern Poland, we collected, during the spring and summer of 2000, more than 1,200 individuals of the ground beetle (Pterostichus oblongopunctatus) to examine the relationship between pollution level and body mass. Animals from one additional sampling in May 2001 were used to measure body caloric value to verify whether metal pollution has an effect on the energy content of the body. The study sites were located in an area with a history of zinc and lead mining and smelting dating back to medieval times. Metal concentrations in the humus layer ranged from 200 to 9,600 mg/kg of zinc, 120 to 1,600 mg/kg of lead, and 3 to 82 mg/kg of cadmium. We found a significant increase in body mass with increasing pollution level. The beetles from all sites collected near the end of the season were lighter. However, no statistically significant trend in body caloric value was detected. We suggest that the high metal tolerance of the species, combined with altered interspecies competition at the polluted sites, is responsible for the positive correlation between soil metal concentration and body mass.

Heavy Metal Accumulation in Plants on Mn Mine Tailings

The Xiangtan Manganese (Mn) Mine in the middle of Hunan Province, China, has been mined since 1913 with mine tailings including excavated wastes, wastewater, and smelting wastes. A survey was conducted on the Mn mine tailing soils and eight plants on the Mn mine tailings. The concentrations of soil Mn, Pb, and Cd and the metal-enrichment traits of these eight plants were analyzed simultaneously. Exceptionally high concentrations of these three metals were found in the soils, especially on the tailing dam. Each plant investigated in this study accumulated the three heavy metals, but no hyperaccumulator of these metals was found. However, analysis indicated that Poa pratensis Linn., Gnaphalium affine D. Don, Pteris vittata L., Conyza canadensis (L.) Cronq., and Phytolacca acinosa Roxb. possessed specially good metal-enrichment and metal-tolerant traits. P. pratensis, G. affine, and P. vittata were Pb-tolerant plants; and C. canadensis, P. pratensis, and G. affine were Cd-tolerant plants. P. acinosa had a great tolerance to Mn, and it was a valuable plant for on-site phytoremediation. Phragmites communis Trin. was found to have high metal tolerance and economic benefit as a raw material for paper and should be considered for soil remediation. G. affine and C. canadensis had excessive accumulation of Mn and could be useful in phytoremediation. However, although P. pratensis was a good accumulator it was not a suitable plant for soil remediation because its biomass was too little.

Response of microbial activities to heavy metals in a neutral loamy soil treated with biosolid

Application of biosolid on land has been widespread in numerous countries for last several decades. This study performed incubation experiments by mixing a neutral loamy soil and biosolid enriched in Cu, Pb and Zn to explore how heavy metal affects soil mineralization and microbial biomass. The experimental results indicated that large nutrient, microorganism and C sources from biosolid were beneficial to microbial respiration. However, compared to the biosolid alone treatment, the supplemented Cu, Pb and Zn in biosolid reduced the mineralized C by roughly 36%. This phenomenon was probably caused by a portion of the Cu, Pb and Zn being complexed with organic matter to prevent decomposition of organic carbon by microorganisms. Equally, soil treated with biosolid increased the quantity of mineralized N by approximately five-fold and accelerated the rate of N mineralization by about one-fold compared to untreated soil. Notably, addition of heavy metals impaired the mineralization process, particularly when Pb reached about 64%. The reduced N mineralization occurred for similar reasons to the microbial respiration. The addition of biosolid in soil considerably increased the amount of mineralizable N; however, the increase was lower in biosolid-treated soil spiked by heavy metals. The addition of heavy metals in the soil–biosolid mixture clearly reduced the microbial biomasses C (MBC) and N (MBN), indicating that the microbial activities had been disrupted by the heavy metals. The microbial biomass C/N ratio had changed initially from 8 to 13 at the end of incubation period, owing to various groups of microbes expressing different mechanisms of metabolism, indicating that the microbial population had changed from bacteria to fungi, which had higher metal tolerance.

Lead and zinc accumulation and tolerance in populations of six wetland plants

Wetland plants such as Typha latifolia and Phragmites australis have been indicated to show a lack of evolution of metal tolerance in metal-contaminated populations. The aim of the present study is to verify whether other common wetland plants such as Alternanthera philoxeroides and Beckmannia syzigachne, also possess the same characteristics. Lead and zinc tolerances in populations of six species collected from contaminated and clean sites were examined by hydroponics. In general, the contaminated populations did not show higher metal tolerance and accumulation than the controls. Similar growth responses and tolerance indices in the same metal treatment solution between contaminated and control populations suggest that metal tolerance in wetland plants are generally not further evolved by contaminated environment. The reasons may be related to the special root anatomy in wetland plants, the alleviated metal toxicity by the reduced rooting conditions and the relatively high innate metal tolerance in some species.