It's a fungi old world

Fungi do more than rot fruit and veg: they have a profound role in geochemistry. Simon Hadlington explains.

We’ve all had that sinking feeling when we reach for a juicy orange from the fruit bowl only to discover that it has a soggy patch of powdery blue-and-white mould growing on it. Fungi are ubiquitous and for obvious reasons are generally seen as agents of rot. And indeed they are fundamentally important in recycling organic matter at both the local and global level.

But fungi play a more subtle role in the cycling of elements. Their activity is not confined to matter made mainly from carbon and nitrogen; as well as rotting down animal and vegetable, they also interact intimately with mineral. Not only do fungi cycle important inorganic components of minerals, including metals, phosphorus and sulphur, they also play a role in breaking down the surfaces of rocks and even materials used in buildings in a process called bioweathering. And the ability of fungi both to solubilise and precipitate a range of metals in the environment (processes termed mobilisation and immobilisation), means that they could be potentially useful in bioremediation - the use of biological agents to clean up contaminated land and effluents.

In the Division of Environmental and Applied Biology at the University of Dundee, UK, Geoffrey Gadd and his colleagues Euan Burford and Marina Fomina are providing new insights into the part that microorganisms, including fungi, play in shaping the mineral world around us in the discipline known as biogeochemistry.

’The most important perceived environmental roles of fungi are as decomposer organisms,’ says Gadd. ’However, a broad appreciation of their role as agents of biogeochemical change is lacking, and apart from obvious connections with the carbon cycle they are frequently neglected within broader microbiological and geochemical spheres.’

While rock substrates can be weathered by the mechanical action of the filamentous hyphae of fungi (growing hyphae can produce enough hydraulic pressure to split rock) chemical weathering is believed to be more important. This is an opinion shared by Gadd, who says that ’microbes, including fungi, can produce chemical weathering of rocks and minerals through excretion of, for example, hydrogen ions, organic acids and metabolites. And even carbon dioxide produced during respiration can lead to carbonic acid attack on mineral surfaces. Biochemical weathering of rocks can result in changes in the micro-topography of minerals through pitting and etching of mineral surfaces, mineral displacement activities and even complete dissolution of mineral grains’.

But how do fungi colonise the surface of rock - a biologically inert and even hostile environment? The answer lies in the presence of other, hardier microbes that can survive in these extreme conditions. Once one particular organism gains a foothold, it provides the nutrients for others to follow. So-called poikilotrophic microorganisms can tolerate extreme micro-climatic conditions of little or no light, extreme pH values and little water. These organisms may also secrete polysaccharides into the environment forming a ’biofilm’, which in turn can be colonised by other organisms, including fungi.

’Inorganic rock substrates do not necessarily favour fungal growth, although the presence of organic and inorganic residues on mineral surfaces or within cracks and fissures may encourage proliferation of fungi and other microbes,’ says Gadd. ’In addition, the waste products of algae and bacterial - or dead cells of these organisms - decaying plant material, dust particles, aerosols and animal faeces can all act as nutrient sources for fungi.’

The range of rock types on which fungi have been found is impressive: limestone, soapstone, marble, granite, sandstone and quartz, for example. Even more remarkably, fungi have been found in extremely harsh environments, such as deserts.

Once established on a rock, fungi generally acidify their immediate environment by excreting protons, organic acids and carbonic acid. Laboratory experiments have shown that alkaline rocks are generally more susceptible to fungal attack than acidic rocks.

Different minerals tend to be attacked by specific groups of microbes, says Gadd. ’Research has demonstrated that siderophore-producing fungi [iron-chelating species]can pit and etch microfractures in samples of olivine and glasses under laboratory conditions, and it has also been shown that both natural and man-made and antique medieval glass can be deteriorated by fungi.’

Fungi attack aluminosilicates and silicates, probably by producing organic acids, inorganic acids, alkalis and complexing reagents, although carbonic acid production may also be important. Burford says that among the most important silicate-attacking fungi are organic acid-producing species. ’For example Aspergillus niger has been shown to degrade olivine, dunite, serpentine, muscovite and feldspar, among others. Penicillium expansum causes extensive degradation of basalt, while Penicillium simplicissimum and Scopulariopsis brevicaulis have been shown to release aluminium from aluminosilicates.’

In some coniferous forests in Europe, weathering of rocks such as feldspars and the granite bedrock has been attributed to the production of oxalic, citric, succinic, formic and malic acids by various fungi. Going into more detail, Burford says that ’fungal hyphal tips have been found to produce micro- to millimolar concentrations of these acids that could effectively dissolve calcium-rich feldspars at rates of up to 0.3 to 30 micrometres a year’.

Fungi can also attack rock surfaces through redox action on mineral constituents such as manganese and iron. ’Desert varnish is an oxidised metal layer a few millimetres thick found on rocks and soils of arid and semi-arid regions and is believed to be of fungal and bacterial origin,’ says Fomina. ’For example, fungi of the Lichenothelia genus can oxidise manganese and iron in metal-bearing minerals such as siderite - FeCO ₃ - and rhodochrosite - MnCO ₃, or from metals absorbed from rainfall or windblown dust and precipitate them as oxides.’ Similarly, the oxidation of Fe(II) and Mn(II) by fungi can cause dark layers, called patinas, to form on glass surfaces.

Fomina explains why limestone is especially vulnerable to bioweathering by fungi, and why certain types of fungi are known to known to colonise and degrade limestone. ’Cavities in limestone provide a major habitat for these organisms, particularly in extreme environments such as cold deserts. The production of organic acids such as oxalic and gluconic acids is believed to play a major role in the biochemical degradation of limestone materials.’

Fungi are also known to degrade sandstone, a phenomenon attributed to the production of acids, including acetic, oxalic, citric, formic and tartaric, among others.

As well as mobilising metals from such rocks, fungi can also act to immobilise metals, that is, precipitate them from solution into a solid form, and this too has an effect on the geochemical profile of an environment. Gadd points out that ’fungal biomass can provide a metal sink through either biosorption to cell walls, pigments and extracellular polysaccharides, intracellular accumulation and sequestration, or precipitation of metal compounds on to hyphae as secondary minerals’. The environment’s pH can influence the binding capacity of the fungal biomass, with a lower pH decreasing the binding capacity for metals such as copper, zinc and cadmium.

Fungi can cause the deterioration of carbonate minerals, such as limestone, dolomite and marble, but can also precipitate carbonates. According to Gadd ’fungal filaments mineralised with calcite [CaCO ₃] together with whewellite [calcium oxalate monohydrate] have been reported in limestone and calcareous soils’. It is entirely possible that fungi play a prominent role in stabilising some limestone rock substrates. ’Calcite formation by fungi may occur through indirect processes via the fungal excretion of oxalic acid and the resulting precipitation of calcium oxalate,’ adds Gadd. ’For example oxalic acid excretion and the formation of calcium oxalate results in the dissolution of the internal pore walls of the limestone matrix, so that the solution becomes enriched in carbonate. During passage of the solution through the pore walls, calcium carbonate re-crystallises and this contributes to the hardening of the material.’

Microbial activity can also make oxalate degrade, transforming it into carbonate, and finally resulting in calcite precipitating inside the pores. This in turn can close the pore system and hardens the parent material.

Metal ions can be reduced to their elemental form by fungal activity. So Ag(I) becomes elemental silver, selenate, Se(VI), and selenite, Se(IV), become elemental selenium, while tellurite, Te(IV), gives elemental tellurium.

’Fungi can acidify their environment by proton efflux via H ⁺-ATPases, maintenance of charge balance or as a result of respiratory carbon dioxide accumulation,’ says Burford. Once the environment is acidified, metals can be released from minerals by a number of routes, such as competition between protons and the metal in the mineral or in a metal-anion complex where, in the latter, the anion is protonated and so releases the free metal cation.

Certain fungi produce carboxylic acids, such as oxalic and citric acid, during their metabolism and these can form stable complexes with a large number of metals. ’Many metal citrates are highly mobile and not readily degraded by microorganisms, and the presence of citric acid in soil may therefore enhance the solubility of metals’, explains Burford. ’Oxalic acid can also act as a leaching agent for metals that form soluble oxalate complexes, including aluminium and iron.’

As well as acids, there are other metabolites that fungi can produce that can bind and remove metals in solution. Polysaccharides and pigments are examples of these metabolites, as well as specific iron-chelating compounds called siderophores. Precisely how ’available’ the metals become will depend on the fate of the complexes in the soil.

’Biomethylation is another way by which metals can become solubilised’, says Fomina, who goes on to explain the process: ’A range of fungi can mediate methylation of metalloids like selenium and tellurium. Methyl groups are transferred to the metal and a given species may transform a number of different elements.’ The methylated metal compounds formed by these processes vary in their solubility, volatility and toxicity. For example, the methylated selenium compounds made in this way are volatile and can be lost to the atmosphere.

Another important process in metal mobilisation is redox transformation - the mobilisation of metals from metal-containing compounds by reduction and oxidation. ’For example, oxidation of metal-complexing dimethylsulfide, dimethylsulfoxide or thiosulfate may increase metal availability if metal sulfates are formed,’ says Fomina. On the other hand, the solubilities of iron and manganese increase on reduction of Fe(II) to Fe(II) and Mn(IV) to Mn(II). Reduction of Hg(II) to Hg(0) results in the diffusion of elemental mercury out of cells.

As well as mobilising metals in the environment, fungi can also immobilise them. Paradoxically, while immobilisation results in a decrease in the external activity of the free metal species, it may shift the equilibrium and promote the release of more metal into the soil from bound or insoluble sources.

One of the most obvious processes that results in metal immobilisation is the binding interaction of metals with the surfaces of cells - a phenomenon called biosorption.

’Fungal cell walls consist of about 80-90 per cent polysaccharide, with glycoproteins and some lipids,’ says Gadd. ’The polysaccharide component includes microcrystalline fibrils of beta-linked polysaccharides, chitin, chitosan and beta-glucans. The chemical properties of the functional groups associated with fungal walls, including carboxyl, amine, hydroxyl, phosphate and sulfhydryl groups, provide the basis for the attraction of metals to cell walls.’ Pigments such as melanin can also strongly bind metals.

Immobilisation of metals and minerals can also occur by precipitation within cells or immediately outside them. This can happen if the organism produces or releases anions such as hydroxyl, sulphate, phosphate or carbonate from mineral substrates that can react with soluble metal cations in the environment.

Oxalate is an important component of many fungal interactions with the mineral environment. Most simple metal oxalates are insoluble. Calcium oxalate is commonly found in soils and leaf litter, occurring as the dihydrate, also known as weddellite, or the monohydrate, called whewellite. Crystalline calcium oxalate is formed by a number of fungi by excretion of oxalate and the precipitation of calcium. In this form, the compound represents an important reservoir of calcium and carbon within ecosystems.

However, fungi might also be useful in mopping up metals and radionuclides in the environment. ’Toxic metals in natural, industrial and agricultural soils are a risk to human health,’ notes Gadd. ’Some of the processes by which fungi interact with metals could have potential for treatment of contaminated land as well as liquid effluents.’

For example the solubilisation of toxic metals could provide a route for their removal from affected soils, whereas immobilisation could enable metal ions to be transformed into insoluble, more chemically inert forms.

’These processes could be used in situ, but for fungi are possibly best suited for use in bioreactors where, for example, immobilised metal can be separated from soil components,’ says Gadd. ’Living or dead fungal biomass and fungal metabolites have been used to remove metal species, compounds and particulates and even organometal compounds from solution. There has also been the use of extracellular ligands excreted by fungi, especially from Aspergillus and Penicillium species, to leach metals such as zinc, copper, nickel and cobalt from a variety of materials, including low-grade mineral ores.’

In addition, some fungi naturally associate with the roots of plants, forming a symbiotic relationship called a mycorrhiza, and these can be involved in the uptake and sequestration of metals by the plant. Such systems have potential for cleaning up contaminated soil in the overall process termed phytoremediation, where the metal is accumulated in plant tissues above the ground. Overall, Gadd suggests that the role of fungi in geochemistry is something that warrants greater attention. ’It is clear that fungi have important biogeochemical roles in the biosphere but are frequently neglected within broader microbiological and geochemical research spheres, in contrast to bacteria,’ he says. ’It is timely to draw attention to ’geomycology’ and the interdisciplinary approach that is necessary to further understanding of the important roles that fungi play in the biogeochemical cycling of elements, the chemical and biological mechanisms that are involved, and their environmental and biotechnological significance.

’To achieve a better understanding of fungal-mineral interactions, the development of experimental methods and techniques that reflect and interrogate environmental conditions more precisely is an urgent need.’

Acknowledgements

Simon Hadlington