F-RISA fungal clones as potential bioindicators of organic and metal contamination in soil.

AIMS: This work has examined the effects of a polycyclic aromatic hydrocarbon and selected toxic metals on fungal populations in a soil microcosm. METHODS AND RESULTS: By using fungal ribosomal intergenic spacer analysis (F-RISA) in combination with real-time PCR quantification, four fungi (D63P2-1, D63C2-1, D21Cu1-1 and D63Pb2-2) with specific primer pairs to each were successfully evaluated for their potential as bioindicators in response to pyrene, copper (Cu) and lead (Pb), supplied singly and in combination. CONCLUSIONS: F-RISA coupled with real-time PCR is a useful approach for the identification of microorganisms with potential as bioindicators of organic and toxic metal contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: These bioindicators could be monitored for their population changes that may indicate pollutant-induced perturbations in a given system.

Pyrene degradation and copper and zinc uptake by Fusarium solani and Hypocrea lixii isolated from petrol station soil.

AIMS: This study aimed to isolate and identify potential polycyclic aromatic hydrocarbon (PAH)-degrading and/or metal-tolerant fungi from PAH-contaminated and metal-contaminated soils. METHODS AND RESULTS: Pyrene-degrading fungi were isolated from contaminated soil and tested for metal (Cu, Zn and Pb) compound solubilization and metal accumulation. Three strains of Fusarium solani and one of Hypocrea lixii were able to degrade more than 60% of initial supplied pyrene (100 mg l(-1)) after 2 weeks. The isolates were grown on toxic metal (Cu, Pb and Zn)-containing media: all isolates accumulated Cu in their mycelia to values ranging from c. 5.9 to 10.4 mmol per kg dry weight biomass. The isolates were also able to accumulate Zn (c. 3.7-7.2 mmol per kg dry weight biomass) from zinc phosphate-amended media. None of the isolates accumulated Pb. CONCLUSIONS: These fungal isolates appear to show promise for use in bioremediation of pyrene or related xenobiotics and removal of copper and zinc from wastes contaminated singly or in combination with these substances. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbial responses to mixed organic and inorganic pollution are seldom considered: this research highlights the abilities of certain fungal strains to interact with both xenobiotics and toxic metals and is relevant to other studies on natural attenuation and bioremediation of polluted sites.

Development and optimization of an 18S rRNA-based oligonucleotide microarray for the fungal order Eurotiales.

AIMS: For identification of members of the fungal order Eurotiales, an 18S rRNA gene-based oligonucleotide microarray was developed and optimized. METHODS AND RESULTS: Eurotiales-specific probes covering most members of the Eurotiales as well as species-specific probes were designed and evaluated with three pure cultures (two fungi from the Eurotiales and one fungus from the Hypocreales). Nearly complete 18S rRNA genes of each reference culture were amplified and fluorescently labelled by random priming. CONCLUSIONS: Positive and negative hybridization results confirmed that the Eurotiales probes tested in this study could correctly identify members of the Eurotiales. The species-specific probes were also capable of species-level detection. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings demonstrate the potential applications of a phylogenetic oligonucleotide microarray approach to characterizing fungal species and populations in environmental and other samples.

Lead mineral transformation by fungi.

Pyromorphite (Pb5(PO4)3Cl), the most stable lead mineral under a wide range of geochemical conditions [1], can form in urban and industrially contaminated soils [2] [3] [4] [5]. It has been suggested that the low solubility of this mineral could reduce the bioavailability of lead, and several studies have advocated pyromorphite formation as a remediation technique for lead-contaminated land [3] [5] [6], if necessary using addition of phosphate [6]. Many microorganisms can, however, make insoluble soil phosphate bioavailable [7] [8] [9] [10], and the solubilisation of insoluble metal phosphates by free-living and symbiotic fungi has been reported [11] [12] [13] [14] [15]. If pyromorphite can be solubilised by microbial phosphate-solubilising mechanisms, the question arises of what would happen to the released lead. We have now clearly demonstrated that pyromorphite can be solubilised by organic-acid-producing fungi, for example Aspergillus niger, and that plants grown with pyromorphite as sole phosphorus source take up both phosphorus and lead. We have also discovered the production of lead oxalate dihydrate by A. niger during pyromorphite transformation, which is the first recorded biogenic formation of this mineral. These mechanisms of lead solubilisation, or its immobilisation as a novel lead oxalate, have significant implications for metal mobility and transfer to other environmental compartments and organisms. The importance of considering microbial processes when developing remediation techniques for toxic metals in soils is therefore emphasised.

An integrated microbial process for the bioremediation of soil contaminated with toxic metals.

Microbially catalyzed reactions, which occur in the natural sulfur cycle, have been integrated in a microbiological process to remove toxic metals from contaminated soils. Bioleaching using sulfuric acid produced by sulfur-oxidizing bacteria was followed by precipitation of the leachate metals as insoluble sulfides by sulfate-reducing bacteria. Metal contaminants including Cd, Co, Cr, Cu, Mn, Ni, and Zn were efficiently leached from an artificially contaminated soil. Mn, Ni, and Zn were the only target elements that were significantly leached from soil minerals. Pb leaching was slow and remained incomplete over a period of 180 days. Mineral components such as Fe, Ca and Mg were also leached but the eventual reduction in soil mass was only approximately 10%. An industrially contaminated soil was also efficiently leached and approximately 69% of the main toxic metals present, Cu, Ni, and Mn, were removed after 175 days. The leachate that resulted from the action of sulfur-oxidizing bacteria on contaminated soil was stripped of metals using an anaerobic bioreactor containing a mixed culture of sulfate-reducing bacteria which precipitated soluble metal species as solid metal sulfides. More than 98% of the metals were removed from solution with the exception of Mn, Ni, and Pb, where 80-90% were removed. The metal content of the resultant effluent liquor was low enough to meet European criteria for discharge into the environment.

Microbial solubilization and immobilization of toxic metals: key biogeochemical processes for treatment of contamination.

Microorganisms play important roles in the environmental fate of toxic metals with a multiplicity of physico-chemical and biological mechanisms effecting transformations between soluble and insoluble phases. Such mechanisms are important components of natural biogeochemical cycles for metals and metalloids with some processes being of potential application to the treatment of contaminated materials. This paper will concentrate on three selected aspects which illustrate the key importance of microorganisms in effecting changes in metal(loid) solubility, namely toxic metal sulfide precipitation by sulfate-reducing bacteria, heterotrophic leaching by fungi, and microbial transformations of metalloids, which includes reduction and methylation. The basic microbiology of these processes is described as well as their environmental significance and use in bioremediation.

Fungal production of citric and oxalic acid: importance in metal speciation, physiology and biogeochemical processes.

The production of organic acids by fungi has profound implications for metal speciation, physiology and biogeochemical cycles. Biosynthesis of oxalic acid from glucose occurs by hydrolysis of oxaloacetate to oxalate and acetate catalysed by cytosolic oxaloacetase, whereas on citric acid, oxalate production occurs by means of glyoxylate oxidation. Citric acid is an intermediate in the tricarboxylic acid cycle, with metals greatly influencing biosynthesis: growth limiting concentrations of Mn, Fe and Zn are important for high yields. The metal-complexing properties of these organic acids assist both essential metal and anionic (e.g. phosphate) nutrition of fungi, other microbes and plants, and determine metal speciation and mobility in the environment, including transfer between terrestrial and aquatic habitats, biocorrosion and weathering. Metal solubilization processes are also of potential for metal recovery and reclamation from contaminated solid wastes, soils and low-grade ores. Such 'heterotrophic leaching' can occur by several mechanisms but organic acids occupy a central position in the overall process, supplying both protons and a metal-complexing organic acid anion. Most simple metal oxalates [except those of alkali metals, Fe(III) and Al] are sparingly soluble and precipitate as crystalline or amorphous solids. Calcium oxalate is the most important manifestation of this in the environment and, in a variety of crystalline structures, is ubiquitously associated with free-living, plant symbiotic and pathogenic fungi. The main forms are the monohydrate (whewellite) and the dihydrate (weddelite) and their formation is of significance in biomineralization, since they affect nutritional heterogeneity in soil, especially Ca, P, K and Al cycling. The formation of insoluble toxic metal oxalates, e.g. of Cu, may confer tolerance and ensure survival in contaminated environments. In semi-arid environments, calcium oxalate formation is important in the formation and alteration of terrestrial subsurface limestones. Oxalate also plays an important role in lignocellulose degradation and plant pathogenesis, affecting activities of key enzymes and metal oxido-reduction reactions, therefore underpinning one of the most fundamental roles of fungi in carbon cycling in the natural environment. This review discusses the physiology and chemistry of citric and oxalic acid production in fungi, the intimate association of these acids and processes with metal speciation, physiology and mobility, and their importance and involvement in key fungal-mediated processes, including lignocellulose degradation, plant pathogenesis and metal biogeochemistry.

Bioremedial potential of microbial mechanisms of metal mobilization and immobilization.

Microorganisms play important roles in the environmental fate of toxic metals and radionuclides with a multiplicity of mechanisms effecting transformations between soluble and insoluble forms. These mechanisms are integral components of natural biogeochemical cycles and are of potential for both in situ and ex situ bioremedial treatment processes for solid and liquid wastes.

Microbial treatment of metal pollution--a working biotechnology?

Some of the main processes that remove, immobilize or detoxify heavy metals and radionuclides in the natural environment result from microbial activities. These activities can be harnessed to clean up toxic metal wastes before they enter the wider environment. To date, the most successful biotechnological processes utilize biosorption and bioprecipitation, but other processes such as binding by specific macromolecules may have future potential. Technologies using these processes are currently used to control pollution from diverse sources, including smelters and mine workings.

Accumulation and intracellular compartmentation of lithium ions in Saccharomyces cerevisiae.

Accumulation of Li+ in Saccharomyces cerevisiae X2180-1B occurred via an apparent stoichiometric relationship of 1:1 (K+/Li+) when S. cerevisiae was incubated in the presence of 5 and 10 mM LiCl for 3 h. Other cellular cations (Mg2+, Ca2+ and Na+) did not vary on Li+ accumulation, although lithium chemistry dictates a degree of similarity to Group I and II metal cations. Compartmentation of Li+ was mainly in the vacuole which accounted for 85% of the Li+ accumulated after a 6-h incubation period. The remainder was located in the cytosol with negligible amounts being bound to cell fragments including the cell wall. Transmission electron microscopy of Li(+)-loaded cells revealed enlarged vacuoles compared with control cells. This asymmetric cellular distribution may therefore enhance tolerance of S. cerevisiae to Li+ and ensure that essential metabolic processes in the cytosol are not disrupted.

Mutants of Saccharomyces cerevisiae defective in vacuolar function confirm a role for the vacuole in toxic metal ion detoxification.

To directly define vacuolar role(s) in metal detoxification, we have examined the responses of vacuole-deficient mutants of Saccharomyces cerevisiae to several potentially toxic metals known to be mainly detoxified in the cytosol (Cu, Cd) or the vacuole (Co, Mn, Ni, Zn). Three mutants, deficient in targeting of vacuolar proteins, were used with JSR18 delta 1 being devoid of any vacuole-like structure while ScVatB and ScVatC were deficient in specific protein subunits of the V-ATPase. The results obtained show that the absence of a vacuole or a functional vacuolar H(+)-ATPase was associated with increased sensitivity and a largely decreased capacity of the vacuole-deficient strains to accumulate Zn, Mn, Co and Ni, confirming an essential role for the vacuole in detoxification of these metals. In addition, the lack of vacuolar involvement in detoxification of Cu and Cd was confirmed since these metals did not exhibit increased toxicity towards the vacuolar mutants nor were there significant differences in Cu or Cd accumulation between parental and mutant strains.

Copper accumulation by sulfate-reducing bacterial biofilms.

Sulfate-reducing bacterial biofilms were grown in continuous culture. When exposed to medium containing 20 or 200 microM Cu, biofilms accumulated Cu. Energy-dispersive X-ray analysis (EDXA) showed that accumulation of Cu occurred in the form of sulfides while EDXA mapping of Cu and S in biofilm sections indicated that they were not uniformly distributed but located in the surface of the biofilm. While the polymer content of biofilm exposed to 20 microM Cu did not appear to increase relative to control Cu-free biofilms, biofilms exposed to 200 microM Cu accumulated carbohydrate and smaller amounts of protein throughout the incubation period. The mechanism of uptake, therefore, appeared to be precipitation of Cu sulfides at the biofilm surface or in the liquid phase followed by entrapment of precipitated Cu sulfide by the exopolymer-enhanced biofilm.

Influence of copper on proton efflux from Saccharomyces cerevisiae and the protective effect of calcium and magnesium.

The inhibitory effect of Cu on glucose-dependent H+ efflux from Saccharomyces cerevisiae was manifest at low (micromolar) concentrations, with the time period between the addition of glucose and commencement of H+ efflux, H+ efflux rate and duration all being affected with increasing Cu concentration (5-100 microM). Ca, at a concentration of 0.5 mM, completely removed the inhibitory effect of Cu at concentrations up to 50 microM and considerably reduced it at higher concentrations (up to 150 microM). Mg exhibited a similar but weaker protective effect against the influence of Cu. The protective effect of Ca against 50 microM Cu was evident at low Ca concentrations (2.5-5 microM), whereas Mg was effective at > or = 50 microM. In order to prevent the inhibitory effect of Cu, it was necessary to add Ca or Mg to the cell suspension before Cu addition. It is concluded that the protective effect of Ca and Mg is mediated by competitive and stabilizing interactions at the cell surface as well as physiological functions of Ca and Mg.

The effect of exogenously-supplied nucleosides and nucleotides and the involvement of adenosine 3':5'-cyclic monophosphate (cyclic AMP) in the yeast mycelium transition of Ceratocystis (= Ophiostoma) ulmi.

The yeast-mycelium transition of Ceratocystis (= Ophiostoma) ulmi (NRRL 6404) was induced by exogenously-supplied nucleosides and nucleotides in defined liquid media. During the yeast-mycelium transition, intracellular adenosine 3':5'-cyclic monophosphate (cAMP) levels increased and maximum levels coincided with maximum germination. This, coupled with findings that the cAMP phosphodiesterase inhibitors, theophylline and caffeine, also induced germination and elevated levels of intracellular cAMP, indicated the involvement of cAMP in the regulation of the yeast-mycelium transition.

Negative fungal chemotropism to toxic metals.

Hyphal growth responses of Geotrichum candidum, Gliocladium roseum, Humicola grisea and Trichoderma viride to Cu and Cd were studied using a simple tessellated agar tile system. Negative chemotropic behaviour of hyphae, which included curling and growth away from metal-containing domains, occurred in all species and with both metals. Both toxic metal and sucrose concentrations in the medium modulated the magnitude of the negative chemotropic effects observed. In general, greater concentrations of metals led to a higher level of negative chemotropism in response to Cu and Cd, which could be reduced with increasing concentrations of sucrose in the medium. This suggests that resource availability affects the ability of these fungi to grow into metal-laden domains.

Solubilisation of some naturally occurring metal-bearing minerals, limescale and lead phosphate by Aspergillus niger.

The ability of the soil fungus Aspergillus niger to tolerate and solubilise seven naturally occurring metal-bearing minerals, limescale and lead phosphate was investigated. A. niger was able to solubilise four of the test insoluble compounds when incorporated into solid medium: cuprite (CuO2), galena (PbS), rhodochrosite (Mn(CO3)x) and limescale (CaCO3). A. niger was able to grow on all concentrations of all the test compounds, whether solubilisation occurred or not, with no reduction in growth rate from the control. In some cases, stimulation of growth occurred, most marked with the phosphate-containing mineral, apatite. Precipitation of insoluble copper and manganese oxalate crystals under colonies growing on agar amended with cuprite and rhodochrosite was observed after 1-2 days growth at 25 degrees C. This process of oxalate formation represents a reduction in bioavailability of toxic cations, and could represent an important means of toxic metal immobilisation of physiological and environmental significance.

The osmotic responses of Saccharomyces cerevisiae in K(+)-depleted medium.

Saccharomyces cerevisiae showed reduced growth in a K(+)-depleted medium but was still capable of synthesizing and accumulating glycerol in the presence of 2.28% (w/v) NaCl; glycerol levels were similar in both K(+)-replete and K(+)-depleted media. The activity of glycerol-3-phosphate dehydrogenase, a key enzyme of glycerol synthesis, was inhibited to a similar extent by exogenous K+ and Na+ although such effects may be modified in vivo due to ionic compartmentation within the vacuole. These results indicate that exogenous K+ does not play a significant role in the initiation of glycerol synthesis in osmotically-stressed yeast.

Enrichment with a polyunsaturated fatty acid enhances the survival of Saccharomyces cerevisiae in the presence of tributyltin.

The toxicity of inorganic metal species towards Saccharomyces cerevisiae has been shown to be markedly dependent on cellular fatty acid composition. In this investigation, the influence of fatty acid supplementation on the toxicity of the lipophilic organometal, tributyltin was investigated. Growth of S. cerevisiae was increasingly inhibited when the tributyltin concentration was increased from 0 to 10 microM. However, the inhibitory effect was partly alleviated by supplementation of the medium with 1 mM linoleate (18:2), a treatment that leads to large-scale incorporation of this polyunsaturated fatty acid (to > 60% of total fatty acids) in yeast membrane lipids. Cells that were previously enriched with 18:2 also showed reduced loss of vitality compared to cells grown in the absence of a fatty acid supplement, when exposed to tributyltin. For example, addition of tributyltin to a concentration of 0.1 microM was associated with an approximate 10% reduction in the H+ efflux activity of 18:2-enriched cells, but a 70% reduction in that of fatty acid-unsupplemented cells. Despite the increased tributyltin resistance of 18:2-enriched S. cerevisiae, the level of cell-associated tributyltin was found to be approximately two-fold higher in these organisms than in fatty acid-unsupplemented cells. These results demonstrate an increased resistance of 18:2-enriched membranes to the direct toxic action(s) of tributyltin. This is in contrast to the previously reported effect of 18:2 enrichment on sensitivity of S. cerevisiae to inorganic metal cations.

Nutritional influence on fungal colony growth and biomass distribution in response to toxic metals.

This work examines nutritional influence on fungal colony growth and biomass distribution in response to toxic metals. In low-substrate solid medium, 0.1 mM Cd, Cu and Zn caused a decrease in radial expansion of both Trichoderma viride and Rhizopus arrhizus. However, as the amount of available carbon source (glucose) increased, the apparent toxicity of the metals decreased. These metals also affected the overall length of the fungal mycelium and branching patterns. In low-nutrient conditions, T. viride showed a decrease in overall mycelial length and number of branches in response to Cu, resulting in an extremely sparsely branched colony. Conversely, although Cd also reduced overall mycelial length to about one-third of the control length, the number of branches decreased only slightly which resulted in a highly branched colony with many aberrant features. Cu and Cd induced similar morphological changes in R. arrhizus. A large-scale mycelial-mapping technique showed that disruption of normal growth by Cu and Cd resulted in altered biomass distribution within the colony. When grown on metal-free low-substrate medium, T. viride showed an even distribution of biomass within the colony with some allocation to the periphery. However, Cu caused most of the biomass to be allocated to the colony periphery, while in the presence of Cd, most biomass was located at the interior of the colony. These results imply that such alterations of growth and resource allocation by Cu and Cd may influence success in locating nutrients as well as survival, and that these metals have individual and specific effects on the growing fungus.

Natural abundance 13C-nuclear magnetic resonance spectroscopic analysis of acyclic polyol and trehalose accumulation by several yeast species in response to salt stress.

Analysis of seventeen yeast strains by 13C-NMR spectroscopy has confirmed the significance of glycerol as the sole osmoregulatory solute under salt-stressed conditions, and has shown arabitol to be present in most of the osmotolerant species. Ribitol was detected in some species, including Debaryomyces hansenii, although ribitol accumulation did not correlate with the osmotic pressure of the medium. Relative amounts of arabitol and ribitol decreased in relation to glycerol when the external osmotic pressure was increased. Trehalose was present during exponential growth of some species.