Role of Oxalic Acid Overexcretion in Transformations of Toxic Metal Minerals by Beauveria caledonica

The fungus Beauveria caledonica was highly tolerant to toxic metals and solubilized cadmium, copper, lead, and zinc minerals, converting them into oxalates. This fungus was found to overexcrete organic acids with strong metal-chelating properties (oxalic and citric acids), suggesting that a ligand-promoted mechanism was the main mechanism of mineral dissolution. Our data also suggested that oxalic acid was the main mineral-transforming agent. Cadmium, copper, and zinc oxalates were precipitated by the fungus in the local environment and also in association with the mycelium. The presence of toxic metal minerals often led to the formation of mycelial cords, and in the presence of copper-containing minerals, these cords exhibited enhanced excretion of oxalic acid, which resulted in considerable encrustation of the cords by copper oxalate hydrate (moolooite). It was found that B. caledonica hyphae and cords were covered by a thick hydrated mucilaginous sheath which provided a microenvironment for chemical reactions, crystal deposition, and growth. Cryo-scanning electron microscopy revealed that mycogenic metal oxalates overgrew parental fungal hyphae, leaving a labyrinth of fungal tunnels within the newly formed mineral matter. X-ray absorption spectroscopy revealed that oxygen ligands played a major role in metal coordination within the fungal biomass during the accumulation of mobilized toxic metals by B. caledonica mycelium; these ligands were carboxylic groups in copper phosphate-containing medium and phosphate groups in pyromorphite-containing medium.     

X-ray absorption spectroscopy (XAS) of toxic metal mineral transformations by fungi

Fungi can be highly efficient biogeochemical agents and accumulators of soluble and particulate forms of metals. This work aims to understand some of the physico-chemical mechanisms involved in toxic metal transformations focusing on the speciation of metals accumulated by fungi and mycorrhizal associations. The amorphous state or poor crystallinity of metal complexes within biomass and relatively low metal concentrations make the determination of metal speciation in biological systems a challenging problem but this can be overcome by using synchrotron-based element-specific X-ray absorption spectroscopy (XAS) techniques. In this research, we have exposed fungi and ectomycorrhizas to a variety of copper-, zinc- and lead-containing minerals. X-ray absorption spectroscopy studies revealed that oxygen ligands (phosphate, carboxylate) played a major role in toxic metal coordination within the fungal and ectomycorrhizal biomass during the accumulation of mobilized toxic metals. Coordination of toxic metals within biomass depended on the fungal species, initial mineral composition, the nitrogen source, and the physiological state/age of the fungal mycelium.     

Organic acids production by white rot Basidiomycetes in the presence of metallic oxides

The purpose of the present work was to determine if selected fungal strains belonging to wood-rotting Basidiomycetes are able to grow on and to solubilize different insoluble oxides in solid media. Twenty-eight strains of white rot fungi were checked for their growth on oxide-amended media (ZnO, CaO, Cu2O). All strains displayed growth on Zn-amended plates but to a different extent, and Cu2O-amended plates turned out to be the most toxic oxide. Most of the tested strains solubilized oxalates and produced noticeable clear zones under the mycelium. These clear zones were tested for the presence of organic acids, the level of which was clearly elevated upon exposure of fungal strains to insoluble oxides. We determined the presence of oxalic, malic, and formic acids, with oxalic acid the predominant one.     

Zinc Phosphate and Pyromorphite Solubilization by Soil Plant-Symbiotic Fungi

Acidolysis, complexolysis and metal accumulation were involved in solubilization of zinc phosphate and pyromorphite by a selection of soil fungi representing ericoid and ectomycorrhizal plant symbionts and an endophytic/entomopathogenic fungus, Beauveria caledonica. Zinc phosphate was much more readily solubilized than pyromorphite. According to the relationship between metal mobilization and pH, acidolysis (protonation) was found to be the major mechanism of both zinc phosphate and pyromorphite dissolution for most of the fungi examined. In general, the more metal tolerant fungal strains yielded more biomass, acidified the medium more and dissolved more of the metal mineral than less tolerant strains. However, B. caledonica 4 excreted a substantial amount of oxalic acid (～ 0.8 mM) in the presence of pyromorphite that coincided with a dramatic increase in lead mobilization providing a clear example of complexolysis. Organic acid excretion by fungi was inter- and intraspecific and was strongly influenced by the presence of the toxic metal minerals. When grown on zinc phosphate or pyromorphite, Hymenoscyphus ericae DGC3(UZ) accumulated the lowest metal concentration, but Thelephora terrestris accumulated the highest metal concentration in the biomass. The ability to accumulate water-soluble lead species, representing mainly cytosolic and vacuolar pools, seemed to be connected with pyromorphite-solubilizing ability. B. caledonica 4, which demonstrated the highest ability to dissolve pyromorphite, accumulated the highest water-soluble fraction and total lead concentration in the mycelium. Generally, isolates with a higher zinc-tolerance accumulated significantly less total zinc from zinc phosphate (including the sum of water-soluble and NaCl-extractable zinc) than non-tolerant strains.     

Exudation-reabsorption in a mycorrhizal fungus, the dynamic interface for interaction with soil and soil microorganisms

 The mycelium of Suillus bovinus slowly absorbed [U-¹⁴C]glucose and other tracers from droplets placed on the cords, translocated them to the peripheral hyphae and exuded them into fluid drops on the hyphal tips. The exudate was characterized by ¹H NMR spectroscopy and by sugar and amino acid analysis. The exuded compounds were mainly carbohydrates and peptides. Acetic acid and oxalic acid were also present in the exudate along with a number of unidentified compounds. Released ions (K, Na, Cl, P, Mg and Ca) were identified by X-ray microanalysis. The mycelium was shown to reabsorb up to 65% of the exuded ¹⁴C compounds in 2 days. Glucose, mannitol, glutamic acid (pH 3.2), and Rb⁺ (as well as other mineral ions) were all readily absorbed by the mycelium, while oxalic acid at pH 4.2 and glutamic acid at pH 6.5 were not. Exudation of fluid droplets on the surface of the hydrophobic mycorrhizal fungus S. bovinus may represent an ecophysiologically important function of the extramatrical hyphae, which provides an interface for interaction with the immediate hyphal environment and its other microorganisms where the peripheral hyphae exchange their photosynthetically derived products for nutrients to be used later by the pine host. We hypothesize that actively absorbed carbohydrates from the root are translocated to the peripheral hyphae along a concentration gradient of sugars and polyols by means of active translocation and diffusion in cell elements and by acropetal water transport in the cord vessels. Accepted 27 April 1999     

Ultrastructural localization of heavy metals in the extraradical mycelium and spores of the arbuscular mycorrhizal fungus Glomus intraradices

Arbuscular mycorrhizal fungi, obligate symbionts of most plant species, are able to accumulate heavy metals, thereby, protecting plants from metal toxicity. In this study, the ultrastructural localization of Zn, Cu, and Cd in the extraradical mycelium and spores of the arbuscular mycorrhizal fungus Glomus intraradices grown in monoxenic cultures was investigated. Zinc, Cu, or Cd was applied to the extraradical mycelium to final concentrations of 7.5, 5.0, or 0.45 mmol/L, respectively. Samples were collected at time 0, 8 h, and 7 days after metal application and were prepared for rapid freezing and freeze substitution. Metal content in different subcellular locations (wall, cytoplasm, and vacuoles), both in hyphae and spores, was determined by energy-dispersive X-ray spectroscopy. In all treatments and fungal structures analysed, heavy metals accumulated mainly in the fungal cell wall and in the vacuoles, while minor changes in metal concentrations were detected in the cytoplasm. Incorporation of Zn into the fungus occurred during the first 8 h after metal addition with no subsequent accumulation. On the other hand, Cu steadily accumulated in the spore vacuoles over time, whereas Cd steadily accumulated in the hyphal vacuoles. These results suggest that binding of metals to the cell walls and compartmentalization in vacuoles may be essential mechanisms for metal detoxification.     

Colonization and bioweathering of monazite by Aspergillus niger: solubilization and precipitation of rare earth elements

Geoactive fungi play a significant role in bioweathering of rock and mineral substrates. Monazite is a phosphate mineral containing the rare earth elements (REE) cerium, lanthanum and neodymium. Little is known about geomicrobial transformations of REE-bearing minerals which are also relevant to REE biorecovery from terrestrial and extra-terrestrial reserves. The geoactive soil fungus Aspergillus niger colonized monazite in solid and liquid growth media without any apparent growth inhibition. In a glucose-minerals salts medium, monazite enhanced growth and mycelium extensively covered rock particle surfaces, probably due to the provision of phosphate and essential trace metals. Teeth-like and pagoda-like etching patterns indicated monazite dissolution, with extensive precipitation of secondary oxalate minerals. Biomechanical forces ensued causing aggressive bioweathering effects by tunnelling, penetration and splitting of the ore particles. High amounts of oxalic acid (~46 mM) and moderate amounts of citric acid (~5 mM) were produced in liquid media containing 2% (wt./vol.) monazite, and REE and phosphate were released. Correlation analysis suggested that citric acid was more effective than oxalic acid in REE mobilization, although the higher concentration of oxalic acid also implied complexant activity, as well as the prime role in REE-oxalate precipitation.     

Lead,cadmium and nickel removal efficiency of white-rot fungus Phlebia brevispora

Widespread of heavy metals contamination has led to several environmental problems. Some biological methods to remove heavy metals from contaminated wastewater are being widely explored. In the present study, the efficiency of a white-rot fungus, Phlebia brevispora to remove different metals (Pb, Cd and Ni) has been evaluated. Atomic absorption spectroscopy of treated and untreated metal containing water revealed that all the metals were efficiently removed by the fungus. Among all the used metals, cadmium was the most toxic metal for fungal growth. Phlebia brevispora removed maximum Pb (97·5%) from 100 mmol l⁻¹ Pb solution, which was closely followed by Cd (91·6%) and Ni (72·7%). Scanning electron microscopic images revealed that the presence of metal altered the morphology and fine texture of fungal hyphae. However, the attachment of metal on mycelia surface was not observed during energy-dispersive X-ray analysis, which points towards the intracellular compartmentation of metals in vacuoles. Thus, the study demonstrated an application of P. brevispora for efficient removal of Pb, Cd and Ni from the metal contaminated water, which can further be applied for bioremediation of heavy metals present in the industrial effluent.     

Isotherm and kinetic models and cell surface analysis for determination of the mechanism of metal sorption by Aspergillus versicolor

The isolate Aspergillus versicolor was obtained from an estuary, which is exposed to metal contamination. It was found to have a good metal tolerance and sorption capacity. Further studies revealed that the rate of metal removal from solution is very rapid in the first 5-10 min, and is favoured by a pH of 6.0. The biosorption data obtained was explained by the Freundlich adsorption isotherm model and followed a pseudo-second order kinetics reaction. The fungus showed a higher accumulation of fatty acids when grown in presence of metals as compared to the mycelium grown in absence of the metal; there was also an increase in the saturation index of fatty acids in presence of Cu(2+) which serves as a protective mechanism for the fungus. Fourier Transform Infrared, scanning electron microscopy and EDAX analysis indicated that metal removal from solution by A. versicolor occurred by a passive adsorption to the fungal cell surface, involving an ion exchange mechanism.     

Transporters of ligands for essential metal ions in plants

Essential metals are required for healthy plant growth but can be toxic when present in excess. Therefore plants have mechanisms of metal homeostasis which involve coordination of metal ion transporters for uptake, translocation and compartmentalization. However, very little metal in plants is thought to exist as free ions. A number of small, organic molecules have been implicated in metal ion homeostasis as metal ion ligands to facilitate uptake and transport of metal ions with low solubility and also as chelators implicated in sequestration for metal tolerance and storage. Ligands for a number of essential metals have been identified and proteins involved in the transport of these ligands and of metal-ligand complexes have been characterized. Here we review recent advances in understanding the role of mugineic acid, nicotianamine, organic acids (citrate and malate), histidine and phytate as ligands for iron (Fe), zinc (Zn), copper (Cu), manganese (Mn) and nickel (Ni) in plants, and the proteins identified as their transporters.     

Fungal degradation of calcium-, lead- and silicon-bearing minerals

The aim of this study was to examine nutritional influence on the ability of selected filamentous fungi to mediate biogenic weathering of the minerals, apatite, galena and obsidian in order to provide further understanding of the roles of fungi as biogeochemical agents, particularly in relation to the cycling of metals and associated elements found in minerals. The impact of three organic acid producing fungi (Aspergillus niger, Serpula himantioides and Trametes versicolor) on apatite, galena and obsidian was examined in the absence and presence of a carbon and energy source (glucose). Manifestation of fungal weathering included corrosion of mineral surfaces, modification of the mineral substrate through transformation into secondary minerals (i.e. crystal formation) and hyphal penetration of the mineral substrate. Physicochemical interactions of fungal metabolites, e.g. H⁺ and organic acids, with the minerals are thought to be the primary driving forces responsible. All experimental fungi were capable of mineral surface colonization in the absence and presence of glucose but corrosion of the mineral surface and secondary mineral formation were affected by glucose availability. Only S. himantioides and T. versicolor were able to corrode apatite in the absence of glucose but none of the fungi were capable of doing so with the other minerals. In addition, crystal formation with galena was entirely dependent on the availability of glucose. Penetration of the mineral substrates by fungal hyphae occurred but this did not follow any particular pattern. Although the presence of glucose in the media appeared to influence positively the mineral penetrating abilities of the fungi, the results obtained also showed that some geochemical change(s) might occur under nutrient-limited conditions. It was, however, unclear whether the hyphae actively penetrated the minerals or were growing into pre-existing pores or cracks.     

Zinc Phosphate Transformations by the Paxillus involutus/Pine Ectomycorrhizal Association

In this research, we investigate zinc phosphate transformations by Paxillus involutus/pine ectomycorrhizas using zinc-resistant and zinc-sensitive strains of the ectomycorrhizal fungus under high- and low-phosphorus conditions to further understand fungal roles in the transformation of toxic metal minerals in the mycorrhizosphere. Mesocosm experiments with ectomycorrhizas were performed under sterile conditions with zinc phosphate localized in cellophane bags: zinc and phosphorus mobilization and uptake by the ectomycorrhizal biomass were analyzed. In the presence of a phosphorus source, an ectomycorrhizal association with a zinc-resistant strain accumulated the least zinc compared to a zinc-sensitive ectomycorrhizal association and non-mycorrhizal plants. Under low-phosphorus conditions, mycorrhizal seedlings infected with the zinc-resistant strain increased the dissolution of zinc phosphate and zinc accumulation by the plant. Extended X-ray absorption fine structure analysis of both mycorrhizal and nonmycorrhizal roots showed octahedral coordination of zinc by oxygen-containing ligands such as carboxylates or phosphate. We conclude that zinc phosphate solubilization and zinc and phosphorus uptake by the association depend on ectomycorrhizal infection, strain of the mycobiont, and the phosphorus status of the matrix.     

Biomineralization of metal-containing ores and concentrates

Biomining is the use of microorganisms to extract metals from sulfide and/or iron-containing ores and mineral concentrates. The iron and sulfide is microbially oxidized to produce ferric iron and sulfuric acid, and these chemicals convert the insoluble sulfides of metals such as copper, nickel and zinc to soluble metal sulfates that can be readily recovered from solution. Although gold is inert to microbial action, microbes can be used to recover gold from certain types of minerals because as they oxidize the ore, they open its structure, thereby allowing gold-solubilizing chemicals such as cyanide to penetrate the mineral. Here, we review a strongly growing microbially-based metal extraction industry, which uses either rapid stirred-tank or slower irrigation technology to recover metals from an increasing range of minerals using a diversity of microbes that grow at a variety of temperatures.     

A Model Sheet Mineral System to Study Fungal Bioweathering of Mica

The general objective of this research was to examine fungal interactions with silicate minerals within the context of their roles in bioweathering. To achieve this, we used muscovite, a phyllosilicate mineral (KAl₂[(OH)₂|AlSi₃O₁₀]), in the form of a mineral sheet model system for ease of experimental manipulation and microscopic examination. It was found that test fungal species successfully colonized and degraded the surface of muscovite sheets in both laboratory and field experiments. After colonization by the common soil fungus Aspergillus niger, a network of hyphae covered the surface of the muscovite, and mineral dissolution or degradation was clearly evidenced by a network of fungal “footprints” that reflected coverage by the mycelium. For natural soil incubations, microorganisms associated with muscovite sheet material included biofilms of fungi and bacteria on the surface, while mineral encrustation or adhesion to microbial structures was also observed. Our results show that muscovite sheet is a good model mineral system for examination of microbial colonization and degradation, and this was demonstrated using laboratory and field systems, providing more evidence for the bioweathering significance of fungal activities in the context of silicate degradation and soil formation and development. The approach is also clearly applicable to other rock and mineral-based substrates and a variety of free-living and symbiotic microbial systems.     

Acid phosphatase activity in Pisolithus arrhizus mycelium treated with cadmium dust

The influence of cadmium dust (containing lead, cadmium, copper, zinc, silicium and other elements) on acid phosphatase activity of Pisolithus arrhizus was observed by means of electron microscopy. Dust-treated mycelium showed increased activity of the enzyme, especially on the surface of the cell wall. There was an increase in abundance of autophagic vacuoles marked by a strong phosphatase reaction. An increase in the number of hyphae with diffuse enzyme activity within the cytoplasm coincided with a decrease of lifespan of the fungus, rapid changes in the mictoplasm stage, earlier closing of the dolipori and presumably the earlier autolysis of cell cytoplasm. Hyphae showing strong autolytic activity were separated from other hyphae by the material deposited within the doliporus and this whole area was devoid at that stage of acid phosphatase activity. The role of the enzyme in the mechanism of resistance to toxic elements is discussed.     

Atomic Force Microscopy of the fungi-mineral interface: applications in mineral dissolution, weathering and biogeochemistry

The interaction between mycorrhizal fungi and minerals is of fundamental importance in affecting the geochemical carbon cycle and CO(2) concentration in the atmosphere, alongside roles in soil creation and the release of nutrients. The symbiosis between the fungi and the plant, supported by photosynthesis in the host plant, has as one of its key features the interfacial zone where mineral and fungi come into contact. At this interface, the organism exudes a complex mixture of organic acids, chelating molecules, protons, and extracellular polysaccharide. In this review, examples will be given of recent Atomic Force Microscopy experiments to monitor the colonization of phyllosilicate minerals in sterile controlled microcosm environments containing only tree seedlings, mineral chips and mycorrhizal fungi. The surface activity of the colonizing fungal hyphae is extensive and complex. In complementary experiments involving exposure of minerals surfaces to single organic acids, it has been possible to monitor dissolution at the unit cell level and to extract activation energies for specific dissolution processes, for example 49kJmol(-1) for 100mM oxalic acid acting upon a biotite sample. The link between these simpler model experiments and the whole microcosm studies is illustrated partly by observations of fungal-colonized mineral surfaces from microcosms after careful removal of the organism and biolayer. These mineral surfaces give clear indications of basal plane modification and fungal weathering.     

Accumulative phases for heavy metals in limnic sediments

Data from mechanical concentrates of recent sediments indicate that clay minerals, clay-rich aggregates and heavy minerals are the major carriers of heavy metals in detrital sediment fractions. Hydrous Fe/Mn oxides and carbonates and sulfides, in their specific environments, are the predominant accumulative phases for heavy metals in autochthonous fractions. Sequential chemical extraction techniques permit the estimation of characteristic heavy metal bonding forms: exchangeable metal cations, easily reducible, moderately reducible, organic and residual metal fractions, whereby both diagenetic processes and the potential availability of toxic compounds can be studied. The data from lakes affected by acid precipitation indicate that zinc, cobalt and nickel are mainly released from the easily reducible sediment fractions and cadmium from organic phases. In contrast at pH 4.4, neither lead nor copper seem to be remobilized to any significant extent. Immobilization by carbonate precipitation seems to provide an effective mechanism for the reduction of dissolved inputs 9f metals such as zinc and cadmium in pH-buffered, hard water systems.     

Bioleaching of spent fluid catalytic cracking catalyst using Aspergillus niger

The use of the fungus Aspergillus niger for the bioleaching of heavy metals from spent catalyst was investigated, with fluid catalytic cracking (FCC) catalyst as a model. Bioleaching was examined in batch cultures with the spent catalysts at various pulp densities (1-12%). Chemical leaching was also performed using mineral acids (sulphuric and nitric acids) and organic acids (citric, oxalic and gluconic acids), as well as a mixture of organic acids at the same concentrations as that biogenically produced. It was shown that bioleaching realised higher metal extraction than chemical leaching, with A. niger mobilizing Ni (9%), Fe (23%), Al (30%), V (36%) and Sb (64%) at 1% pulp density. Extraction efficiency generally decreased with increased pulp density. Compared with abiotic controls, bioleaching gave rise to higher metal extractions than leaching using fresh medium and cell-free spent medium. pH decreased during bioleaching, but remained relatively constant in both leaching using fresh medium and cell-free spent medium, thus indicating that the fungus played a role in effecting metal extraction from the spent catalyst.     

High resolution characterization of ectomycorrhizal fungal-mineral interactions in axenic microcosm experiments

Microcosms with Pinus sylvestris seedlings in symbiosis with the fungus mycorrhizal Paxillus involutus were established, and atomic force microscopy (AFM) was used to characterise plant photosynthate-driven fungal interactions with mineral surfaces. Comparison of images of the same area of the minerals before and after mycorrhizal fungal colonization showed extensive growth of hyphae on three different mineral surfaces – hornblende, biotite and chlorite. A layer of biological exudate, or biolayer, covered the entire mineral surface and was composed of globular features of diameter 10–80 nm, and the morphology of the biolayer differed among mineral types. Similar-sized components were found on the fungal hyphae, but with a more elongated profile. Biolayer and hyphae surfaces both appeared to be hydrophobic with the hyphal surfaces yielding higher maximal adhesive interactions and a wider range of values: the mean (± SE) adhesive forces were 2.63 ± 0.03 and 3.46 ± 0.18 nN for biolayer and hypha, respectively. The highest adhesion forces are preferentially localized at the hyphal surface above the Spitzenkörper region and close to the tip, with a mean interaction force in this locality of 5.24 ± 0.49 nN. Biolayer thickness was between 10 and 40 nm. The underlying mineral was easily broken up by the tip, in contrast to the native mineral. These observations of mineral surfaces colonised by mycorrhizal fungus demonstrate how fungal hyphae are able to form a layer of organic exudates, or biolayer, and its role in hyphal attachment and potential weathering of ferromagnesian silicates, which may supply nutrients to the plant.     

A glass fiber filter technique for studying nutrient uptake by fungi: The technique used on colonies grown on nutrient gradients of carbon and phosphorus

In most natural environments supporting fungal growth, nutrients are heterogeneously distributed in space. Growth of a fungus will thus take place in an environment characterized by gradients. A system has been developed for growth of fungi on opposing carbon and mineral nutrient gradients, present in liquid medium in glass fiber filters. By labeling the carbon or phosphorus in the medium, the amount of carbon or phosphorus accumulated inside hyphae ofRhizopus nigricans and the amount still present outside the hyphae were determined. The distribution of labeled C and P in the medium and in the colonies of the fungus grown in the presence and absence of initial gradients in the medium was compared. In both cases, little carbon or phosphorus was found remaining in the medium after fungal growth. With colonies grown on media without an initial gradient, two peaks of carbon and phosphorus accumulation were found, but when there was a gradient there was only one such peak. These peaks coincided with the regions of greatest sporulation. It was concluded, by comparing the distribution of the total amount of carbon inside and outside mycelium grown on gradients with that in control media which was uninoculated, that the translocation of carbon inside the mycelium could have been brought about by simple diffusion.