Fungal Bioweathering of Mimetite and a General Geomycological Model for Lead Apatite Mineral Biotransformations

Fungi play important roles in biogeochemical processes such as organic matter decomposition, bioweathering of minerals and rocks, and metal transformations and therefore influence elemental cycles for essential and potentially toxic elements, e.g., P, S, Pb, and As. Arsenic is a potentially toxic metalloid for most organisms and naturally occurs in trace quantities in soil, rocks, water, air, and living organisms. Among more than 300 arsenic minerals occurring in nature, mimetite [Pb₅(AsO₄)₃Cl] is the most stable lead arsenate and holds considerable promise in metal stabilization for in situ and ex situ sequestration and remediation through precipitation, as do other insoluble lead apatites, such as pyromorphite [Pb₅(PO₄)₃Cl] and vanadinite [Pb₅(VO₄)₃Cl]. Despite the insolubility of mimetite, the organic acid-producing soil fungus Aspergillus niger was able to solubilize mimetite with simultaneous precipitation of lead oxalate as a new mycogenic biomineral. Since fungal biotransformation of both pyromorphite and vanadinite has been previously documented, a new biogeochemical model for the biogenic transformation of lead apatites (mimetite, pyromorphite, and vanadinite) by fungi is hypothesized in this study by application of geochemical modeling together with experimental data. The models closely agreed with experimental data and provided accurate simulation of As and Pb complexation and biomineral formation dependent on, e.g., pH, cation-anion composition, and concentration. A general pattern for fungal biotransformation of lead apatite minerals is proposed, proving new understanding of ecological implications of the biogeochemical cycling of component elements as well as industrial applications in metal stabilization, bioremediation, and biorecovery.     

Fungal biotransformation of zinc silicate and sulfide mineral ores

In this work, several fungi with geoactive properties, including Aspergillus niger, Beauveria caledonica and Serpula himantioides, were used to investigate their potential bioweathering effects on zinc silicate and zinc sulfide ores used in zinc extraction and smelting, to gain understanding of the roles that fungi may play in transformations of such minerals in the soil, and effects on metal mobility. Despite the recalcitrance of these minerals, new biominerals resulted from fungal interactions with both the silicate and the sulfide, largely resulting from organic acid excretion. Zinc oxalate dihydrate was formed through oxalate excretion by the test fungi and the mineral surfaces showed varying patterns of bioweathering and biomineral formation. In addition, calcium oxalate was formed from the calcium present in the mineral ore fractions, as well as calcite. Such metal immobilization may indicate that the significance of fungi in effecting metal mobilization from mineral ores such as zinc silicate and zinc sulfide is rather limited, especially if compared with bacterial sulfide leaching. Nevertheless, important bioweathering activities of fungi are confirmed which could be of local significance in soils polluted by such materials, as well as in the mycorrhizosphere.     

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.     

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.     

Biotransformation of struvite by Aspergillus niger: phosphate release and magnesium biomineralization as glushinskite

Struvite (magnesium ammonium phosphate-MgNH₄PO₄·6H₂O), which can extensively crystallize in wastewater treatments, is a potential source of N and P as fertilizer, as well as a means of P conservation. However, little is known of microbial interactions with struvite which would result in element release. In this work, the geoactive fungus Aspergillus niger was investigated for struvite transformation on solid and in liquid media. Aspergillus niger was capable of solubilizing natural (fragments and powder) and synthetic struvite when incorporated into solid medium, with accompanying acidification of the media, and extensive precipitation of magnesium oxalate dihydrate (glushinskite, Mg(C₂O₄).2H₂O) occurring under growing colonies. In liquid media, A. niger was able to solubilize natural and synthetic struvite releasing mobile phosphate (PO₄³⁻) and magnesium (Mg²⁺), the latter reacting with excreted oxalate resulting in precipitation of magnesium oxalate dihydrate which also accumulated within the mycelial pellets. Struvite was also found to influence the morphology of A. niger mycelial pellets. These findings contribute further understanding of struvite solubilization, element release and secondary oxalate formation, relevant to the biogeochemical cycling of phosphate minerals, and further directions utilizing these mechanisms in environmental biotechnologies such as element biorecovery and biofertilizer applications.     

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°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.     

Uranium phosphate biomineralization by fungi

Geoactive soil fungi were investigated for phosphatase‐mediated uranium precipitation during growth on an organic phosphorus source. Aspergillus niger and Paecilomyces javanicus were grown on modified Czapek–Dox medium amended with glycerol 2‐phosphate (G2P) as sole P source and uranium nitrate. Both organisms showed reduced growth on uranium‐containing media but were able to extensively precipitate uranium and phosphorus‐containing minerals on hyphal surfaces, and these were identified by X‐ray powder diffraction as uranyl phosphate species, including potassium uranyl phosphate hydrate (KPUO₆.3H₂O), meta‐ankoleite [(K_1.7Ba_0.2)(UO₂)₂(PO₄)₂.6H₂O], uranyl phosphate hydrate [(UO₂)₃(PO₄)₂.4H₂O], meta‐ankoleite (K(UO₂)(PO₄).3H₂O), uramphite (NH₄UO₂PO₄.3H₂O) and chernikovite [(H₃O)₂(UO₂)₂(PO₄)₂.6H₂O]. Some minerals with a morphology similar to bacterial hydrogen uranyl phosphate were detected on A. niger biomass. Geochemical modelling confirmed the complexity of uranium speciation, and the presence of meta‐ankoleite, uramphite and uranyl phosphate hydrate between pH 3 and 8 closely matched the experimental data, with potassium as the dominant cation. We have therefore demonstrated that fungi can precipitate U‐containing phosphate biominerals when grown with an organic source of P, with the hyphal matrix serving to localize the resultant uranium minerals. The findings throw further light on potential fungal roles in U and P biogeochemistry as well as the application of these mechanisms for element recovery or bioremediation.     

Monazite transformation into Ce- and La-containing oxalates by Aspergillus niger

Monazite is a naturally occurring lanthanide (Ln) phosphate mineral [Ln _x(PO₄) _y] and is the main industrial source of the rare earth elements (REE), cerium and lanthanum. Endeavours to ensure the security of supply of elements critical to modern technologies view bioprocessing as a promising alternative or adjunct to new methods of element recovery. However, relatively little is known about microbial interactions with REE. Fungi are important geoactive agents in the terrestrial environment and well known for properties of mineral transformations, particularly phosphate solubilization. Accordingly, this research examined the capability of a ubiquitous geoactive soil fungus, Aspergillus niger, to affect the mobility of REE in monazite and identify possible mechanisms for biorecovery. It was found that A. niger could grow in the presence of monazite and mediated the formation of secondary Ce and La-containing biominerals with distinct morphologies including thin sheets, orthorhombic tablets, acicular needles, and rosette aggregates which were identified as cerium oxalate decahydrate (Ce₂(C₂O₄)₃·10H₂O) and lanthanum oxalate decahydrate (La₂(C₂O₄)₃·10H₂O). In order to identify a means for biorecovery of REE via oxalate precipitation the bioleaching and bioprecipitation potential of biomass-free spent culture supernatants was investigated. Although such indirect bioleaching of REE was low from the monazite with maximal lanthanide release reaching >40 mg L⁻¹, leached REE were efficiently precipitated as Ce and La oxalates of high purity, and did not contain Nd, Pr and Ba, present in the original monazite. Geochemical modelling of the speciation of oxalates and phosphates in the reaction system confirmed that pure Ln oxalates can be formed under a wide range of chemical conditions. These findings provide fundamental knowledge about the interactions with and biotransformation of REE present in a natural mineral resource and indicate the potential of oxalate bioprecipitation as a means for efficient biorecovery of REE from solution.     

A new insight into lead (II) tolerance of environmental fungi based on a study of Aspergillus niger and Penicillium oxalicum

Environmental microorganisms have been widely applied in heavy metal remediation. This study explored the mechanisms of lead tolerance of two typical filamentous fungi, Aspergillus niger and Penicillium oxalicum. It is shown that the mechanisms of reducing Pb toxicity by these two fungi have three major pathways. The secreted oxalic acid can react with Pb (II) to form insoluble Pb minerals, primarily lead oxalate. Then, the enhanced biosorption via forming new border of cell wall prevents the transportation of Pb (II) into hypha. In addition, the fungal activity could be maintained even at high Pb concentration due to the intracellular accumulation. It was confirmed that A. niger has the higher Pb tolerance (up to 1500 mg l⁻¹ Pb level) compared with P. oxalicum (up to 1000 mg l⁻¹). Meanwhile, Pb levels below 1000 mg l⁻¹ partially stimulate the bioactivity of A. niger, which was confirmed by its elevated respiration (from 53 to 63 mg C l⁻¹ medium h⁻¹). This subsequently enhanced microbial functions of A. niger to resist Pb toxicity. A better understanding of Pb tolerance of these two fungi sheds a bright future of applying them to remediate lead-contaminated environments.     

Fungal transformation of natural and synthetic cobalt-bearing manganese oxides and implications for cobalt biogeochemistry

Manganese oxide minerals can become enriched in a variety of metals through adsorption and redox processes, and this forms the basis for a close geochemical relationship between Mn oxide phases and Co. Since oxalate-producing fungi can effect geochemical transformation of Mn oxides, an understanding of the fate of Co during such processes could provide new insights on the geochemical behaviour of Co. In this work, the transformation of Mn oxides by Aspergillus niger was investigated using a Co-bearing manganiferous laterite, and a synthetic Co-doped birnessite. A. niger could transform laterite in both fragmented and powder forms, resulting in formation of biomineral crusts that were composed of Mn oxalates hosting Co, Ni and, in transformed laterite fragments, Mg. Total transformation of Co-doped birnessite resulted in precipitation of Co-bearing Mn oxalate. Fungal transformation of the Mn oxide phases included Mn(III,IV) reduction by oxalate, and may also have involved reduction of Co(III) to Co(II). These findings demonstrate that oxalate-producing fungi can influence Co speciation in Mn oxides, with implications for other hosted metals including Al and Fe. This work also provides further understanding of the roles of fungi as geoactive agents which can inform potential applications in metal bioremediation, recycling and biorecovery.     

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.     

Rock phosphate solubilization by abiotic and fungal-produced oxalic acid: reaction parameters and bioleaching potential

Oxalic acid-producing fungi play an important role in biogeochemical transformations of rocks and minerals and possess biotechnological potential for extraction of valuable elements from primary or waste ores and other solid matrices. This research investigates the extraction of phosphate from rock phosphate (RP) by oxalic acid. Reaction parameters were derived using pure oxalic acid solutions to solubilize RP. It was found that the oxalic acid concentration was the main factor driving reaction kinetics. Excess oxalic acid could retard the reaction due to calcium oxalate encrustation on RP surfaces. However, complete P extraction was reached at stoichiometric proportions of apatite and oxalic acid. This reaction reached completion after 168 h, although most of the P (up to 75%) was released in less than 1 h. Most of the Ca released from the apatite formed sparingly soluble calcium oxalate minerals, with a predominance of whewellite over weddellite. Bioleaching of RP employing biomass-free spent culture filtrates containing oxalic acid (100 mM) produced by Aspergillus niger extracted ~ 74% of the P contained in the RP. These findings contribute to a better understanding of the reaction between apatite and oxalic acid and provide insights for potential applications of this process for biotechnological production of phosphate fertilizer.     

Colonization,penetration and transformation of manganese oxide nodules by Aspergillus niger

In this study, the ability of the geoactive fungus Aspergillus niger to colonize and transform manganese nodules from the Clarion-Clipperton Zone in both solid and liquid media was investigated. Aspergillus niger was able to colonize and penetrate manganese nodules embedded in solid medium and effect extensive transformation of the mineral in both fragmented and powder forms, precipitating manganese and calcium oxalates. Transformation of manganese nodule powder also occurred in a liquid medium in which A. niger was able to remove the fine particles from suspension which were accumulated within the central region of the resulting mycelial pellets and transformed into manganese oxalate dihydrate (lindbergite) and calcium oxalate dihydrate (weddellite). These findings contribute to an understanding of environmental processes involving insoluble manganese oxides, with practical relevance to chemoorganotrophic mineral bioprocessing applications, and, to the best of our knowledge, represent the first demonstration of fundamental direct and indirect interactions between geoactive fungi and manganese nodules.     

Solubilization of Magnesium-Bearing Silicate Minerals and the Subsequent Formation of Glushinskite by Aspergillus niger

Microbes may play a substantial role in the weathering and alteration of minerals. However, not enough concerns have been realized about the complexity of microbe-mineral interactions. The present work reports the interactions between fungi and minerals with emphasis on the role of silicate minerals as the metal donor for the precipitation of secondary mineral. Herein, two magnesium-bearing silicate minerals with different structures, forsterite and talc, were added to the submerged cultures of Aspergillus niger (A. niger). It is shown that forsterite exhibits a better solubilization effect than talc, and the secondary mineral glushinskite only precipitates in the presence of forsterite substrates. Oxalic acid excreted by A. niger plays a key role in the biological leaching and mineralization processes. Moreover, the forsterite particles with certain size added to the cultures tend to inhibit the aggregation of fungal biomass, and therefore affect the morphology of A. niger aggregates in the submerged culture. With varying forsterite particle size, distinct fungal morphological forms including mycelial pellets and freely dispersed hyphae can be formed, which have a direct impact on fungal metabolism and ultimately result in varied crystallization habits of the neo-minerals. Particularly in the culture with ?40/+60 mesh forsterite particles, the mineralized glushinskite mainly exhibit tubular forms; whereas when forsterite particles were at ?100/+120 mesh or ?200 mesh, pyramidal prisms are obtained. We show that the environmental factors such as the particle size of mineral substrate could influence the fungal morphology and metabolic activities, thereby leading to different morphological neo-minerals. The dependence of biomineral morphology on the environmental factors can open up a novel avenue to understanding the microorganism-environment interactions.     

Sr Isotope Tracing of Differences in the Effects of Microorganisms on Weathering of Calcite and Apatite

To study differences in the effects of microorganisms on weathering of calcite and apatite, one strain of Aspergillus niger (A. niger) and one strain of Penicillium glaucum (P. glaucum), which respectively contain the mixture of calcite and apatite were cultivated for 24 days in the sucrose-potato culture medium, supernatant was taken every three days from the culture medium, followed by the determination of Ca²⁺ and Sr²⁺ contents and Sr isotopic ratios. The results of measurement showed that the Sr isotope ratios in the supernatant from the culture medium are intermediate between those of the end-member constituents calcite and apatite (0.70721-0.70861). Results of isotope mixing equations to calculation showed that in the first 15 days A. niger played a dominant role in weathering of calcite in the apatite/calcite mixture. The contribution rate of apatite for Ca²⁺ in the solution increased from 39.0% on the 18th day to 61.6% on the 24th day; P. glaucum played a key role in weathering of apatite in the first 3 days. Ions dissolved from apatite account for 73.9% of the total. It is known from the results of Sr isotope tracing that in the prior period of fungus cultivation A. niger plays a key role in weathering of calcite while P. glaucum plays a key role in weathering of apatite. The ability of P. glaucum to weather calcite tends to intensify progressively over time. Therefore, Sr isotope tracing can be used to accurately recognize differences in the effects of microorganisms on weathering of minerals.     

Fungi,bacteria and soil pH: the oxalate–carbonate pathway as a model for metabolic interaction

The oxalate–carbonate pathway involves the oxidation of calcium oxalate to low‐magnesium calcite and represents a potential long‐term terrestrial sink for atmospheric CO ₂. In this pathway, bacterial oxalate degradation is associated with a strong local alkalinization and subsequent carbonate precipitation. In order to test whether this process occurs in soil, the role of bacteria, fungi and calcium oxalate amendments was studied using microcosms. In a model system with sterile soil amended with laboratory cultures of oxalotrophic bacteria and fungi, the addition of calcium oxalate induced a distinct pH shift and led to the final precipitation of calcite. However, the simultaneous presence of bacteria and fungi was essential to drive this pH shift. Growth of both oxalotrophic bacteria and fungi was confirmed by qPCR on the frc (oxalotrophic bacteria) and 16S rRNA genes, and the quantification of ergosterol (active fungal biomass) respectively. The experiment was replicated in microcosms with non‐sterilized soil. In this case, the bacterial and fungal contribution to oxalate degradation was evaluated by treatments with specific biocides (cycloheximide and bronopol). Results showed that the autochthonous microflora oxidized calcium oxalate and induced a significant soil alkalinization. Moreover, data confirmed the results from the model soil showing that bacteria are essentially responsible for the pH shift, but require the presence of fungi for their oxalotrophic activity. The combined results highlight that the interaction between bacteria and fungi is essential to drive metabolic processes in complex environments such as soil.     

Purification of bioactive natural product against human microbial pathogens from marine sea weed <Emphasis Type="Italic">Dictyota acutiloba</Emphasis> J. Ag.

Herbal extracts play an essential role in treating various diseases. The threats in drug resistant pathogenic microbial strains can be prevented by the un-tapped medicinal principles from plants. The present study has been focused to search for powerful antimicrobial natural products from Dictyota acutiloba J. Ag. against human enteric pathogens and dermatophytic fungi. Chloroform and acetone extracts of Dictyota acutiloba exhibited antimicrobial activity against methicillin resistant Staphylococcus aureus (MRSA), methicillin susceptible Staphylococcus aureus (MSSA), Enterobacter sp., Pseudomonas aeruginosa MTCC741, Salmonella typhi MTCC733, Bacillus subtilis, Klebsiella pneumoniae MTCC109, Candida albicans and Aspergillus niger MTCC281. Purified compounds A₁ and C₁ by column chromatography, TLC and HPLC inhibited the gram positive, gram negative bacteria and fungi. MIC of C₁ and A₁ ranged between 0.5 and 0.9 μg ml⁻¹. The absorption maximum of C₁ and A₁ was 355 nm. Structural characterization of these purified molecules can lead to the new therapeutic molecule to fight the pathogenic microorganisms.     

Plant-driven weathering of apatite - the role of an ectomycorrhizal fungus

Ectomycorrhizal (EcM) fungi are increasingly recognized as important agents of mineral weathering and soil development, with far‐reaching impacts on biogeochemical cycles. Because EcM fungi live in a symbiotic relationship with trees and in close contact with bacteria and archaea, it is difficult to distinguish between the weathering effects of the fungus, host tree and other micro‐organisms. Here, we quantified mineral weathering by the fungus Paxillus involutus, growing in symbiosis with Pinus sylvestris under sterile conditions. The mycorrhizal trees were grown in specially designed sterile microcosms in which the supply of soluble phosphorus (P) in the bulk media was varied and grains of the calcium phosphate mineral apatite mixed with quartz, or quartz alone, were provided in plastic wells that were only accessed by their fungal partner. Under P limitation, pulse labelling of plants with ¹⁴CO₂ revealed plant‐to‐fungus allocation of photosynthates, with 17 times more ¹⁴C transferred into the apatite wells compared with wells with only quartz. Fungal colonization increased the release of P from apatite by almost a factor of three, from 7.5 (±1.1) × 10^?10 mol m^?2 s^?1 to 2.2 (±0.52) × 10^?9 mol m^?2 s^?1. On increasing the P supply in the microcosms from no added P, through apatite alone, to both apatite and orthophosphate, the proportion of biomass in roots progressively increased at the expense of the fungus. These three observations, (i) proportionately more plant energy investment in the fungal partner under P limitation, (ii) preferential fungal transport of photosynthate‐derived carbon towards patches of apatite grains and (iii) fungal enhancement of weathering rate, reveal the tightly coupled plant–fungal interactions underpinning enhanced EcM weathering of apatite and its utilization as P source.