FungiasGeologicAgents - Jagiellonian Universityeko.uj.edu.pl/mycorrhiza/MKBL_2013/konw/10_grzyby jako... · 2013-11-19 · FungiasGeologicAgents 103 fungalgrowth.Interestingly,Manolietal.(1997)foundthatchitin,themajorcompoundof

Geomicrobiology Journal, 17:97–124, 2000Copyright C° 2000 Taylor & Francis0149-0451/00 $12.00 + .00

Fungi as Geologic Agents

KATJA STERFLINGER

Carl von Ossietzky UniversityOldenburg, Germany

Although many studies on fungi and geological processes have been published in recentyears, books and congress proceedings on geomicrobiology focus mainly on prokaryotesand algae. Therefore, it is theaim of this reviewto summarize data on the fungal impact ongeological processes. These processes include the alteration and weathering of rock andminerals, the accumulation of metals, and the conversion of fossil organic carbon. Fossilrecords and fungi in subsurface environments are also discussed. This article especiallyemphasizes the role of epi- and endolithic black meristematic fungi, discussing theirdeteriorative potential on rock as well as their taxonomy and phylogeny. Moreover, theimpact of fungi on weathering of monuments and building materials is describedand newmethods to study fungi-material interactions are presented. The data summarized hereshow that “geomycology” is a highly interesting discipline in view of basic geologicalresearch, as well as biotechnological application.

Basic geological processes include the accumulation of inorganic material, the dispersionof material by solubilization and lique� cation, and the fractionation of substances (Ehrlich1981, 1999). Geomicrobiology also includes the studies of energy fluxes between the livingand the nonliving matter of the earth and is therefore coupled to studies of ecosystems inthe sense of Lindemann (1942) as “the circulation, transformation, and accumulation ofenergy and material with living beings and their activities.” Without doubt, the existenceof the earth in its actual appearance has been considerably influenced if not determined by3.8 billion years of life and its interaction with the nonliving material. The photosyntheticactivity of cyanobacteria changed the atmosphere from the reduced state to the oxidizedstate we know today. Microorganisms recycle carbonate, nitrogen, phosphorus, sulfur, andmany other elements essential for life of macroorganisms on earth. Sediment rocks are theproduct of direct and indirect modes of carbonate precipitation. Even the plate tectonicsand the albedo of the earth are considered to be influenced by organisms (Lovelock 1988;Westbroek and de Bruyn 1996).

Without doubt, life on earth was long dominated by microbial mats, consisting ofmainly prokaryotic organisms such as cyano- and archaebacteria. Today, a wide range ofphotoautotrophic, chemolithotrophic, and chemoorganotrophic bacteria have been investi-gated with respect to their potential to alter, solubilize, and accumulate minerals (Ehrlich1981; Ban� eld and Nealson 1997). In contrast, fungi are still a neglected group with re-spect to exploration of their geomicrobiological potential. Except for lichens, publicationson geomicrobiology including fungi are relatively rare; often, this highly interesting andphysiologically as well as morphologically diverse kingdom is only mentioned as “existing”

Received 4 August 1999; accepted 15 November 1999.Wolfgang E. Krumbein is acknowledged for valuable discussions on the topic, for his support of my work,

and for correction of the manuscript. Sybren de Hoog was a great support for my work on fungal taxonomy. I alsoexpress my thanks to the colleagues in the Centraalbureau voor Schimmelcultures (Baarn, Netherlands) and at thelibrary of the Universitat fur Bodenkultur (Vienna, Austria) for their kind help with the literature search.

Address correspondence to Katja Sterflinger, Carl von Ossietzky University, ICBM, Geomicrobiology, POBox 2503, D-26111 Oldenburg, Germany. E-mail: [email protected]

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98 K. Sterflinger

in microbial communities. Geomicrobiology, in fact, is settled between two disciplines: Inbacteriology, there is more tradition to study the interactions among bacteria and with theirenvironment, whereas in mycology, such studies are still treated as a specialty of botanists,and taxonomy and the ecology of the individual species are given more focus. Questionsstill to be explored include these: (1) Do fungi considerably influence the genesis and di-agenesis of sediments, rocks, and minerals? (2) Do they influence soil formation? (3) Arethey involved in the genesis and conversion of fossil organic carbon?

In 1966, Wolfgang E. Krumbein was one of the � rst geomicrobiologists who repeatedlyhinted that fungi could influence the weathering of rock. He was the � rst to recognize blackfungal colonies on marble as the most detrimental microflora on Mediterranean monuments(Krumbein and Urz õ 1993). Subsequent to Krumbein’s initiative, the black fungi have at-tracted much attention by mycologists. The black fungi now are regarded as the real special-ists of rock decay in arid and semiarid environments. Because the taxonomy and ecology ofthis group are rather new in the � eld of mycology, they are described in more detail in thisreview. The influx of lichenized fungi on the weathering of rocks is quite well investigatedand numerous publications exist on the topic (Easton 1994; Gehrmann et al. 1992; seePiervittori et al. [1994] for a literature list); therefore, lichens will not be treated here.

Fungal Attack on Minerals

For the attackof mineral compounds, two basicmechanisms are described that can be carriedout by both fungi and bacteria: (1) acid attackby organic and inorganic acids, (2) mechanicalactivities that disaggregate or aggregate particles and thus decrease or increase the reactivesurface. In the following sections, the specialties of fungi are described.

Alteration of Carbonates

Boring of carbonates has long been assigned almost exclusively to the mechanical andchemical activities of either animals (Haigler 1969; Riedl 1966), bryophytes (Fuxing et al.1993), lichens (Zahlbruckner 1890; Fry 1927; Mellor 1923; Gehrmann et al. 1992), or algaeand cyanobacteria (Danin et al. 1982; Golubic et al. 1975; Schneider 1976) even thoughfungi boring carbonate shells were reported by Bornet and Flahaut in 1889. Fungi were � rstconsidered to be agents of carbonate deterioration by Krumbein (1969). An extensive studywas carried out by Braams (1992), who has isolated from sandstone surfaces in Germany 70species of hyphomycetes that attack the rock by excretion of acetic, oxalic, citric, formic,fumaric, glyoxylic, gluconic, and to a lesser extent, tartaric acid (Table 1, Figure 1A, B). Inaddition to the species belonging to genera of the typical soil flora Penicillium,Trichoderma,and Cladosporium , one of the most abundant species found was the black yeast Exophialajeanselmei. This � nding led to a more intensive search for the black yeast-like fungi on rocksurfaces because these were formerly known only as plant- or human-associated organisms(de Hoog and Hermanides-Nijhof 1977). A compilation of fungi on rock is given in Table 1along with information about their acid production and pigmentation. The � rst feature isimportant for the solubilization of minerals; the latter, for the biogenic patination of rock,which is described in more detail in the section on weathering of monuments and buildings.

Braams (1992) reported that the growth of fungi on rock might be limited by thepresence of clay minerals. However, this is not true for all fungi. For example, Epicoccumpurpurascens, which frequently inhabits rock surfaces in urban environments (Sterflinger1999), is favored by the presence of clay minerals (Filip et al. 1972).

Especially in hot and cold deserts, rock flora is dominated by a special group of darklypigmented fungi that form clump-like colonies epi- and endolithically in limestone, marble,

Fungi as Geologic Agents 99

FIGURE 1 (A) SEM micrograph of fungal hyphae attached to mineral particles of a sand-stone surface. (B) Etching of a calcium carbonate crystal by a fungal hyphae. (C) Fungalgrowth between the crystals of quartz dissolves the carbonatic binding matrix and results ingranular disintegration (bar = 800 l m). (D) Growth margin of a self-overgrowing bryozoanspecies attacked by fungal mycelium. The left side shows the older part of the bryozoancolony being decayed by the fungi; the right part shows the new bryozoan layer (photo:Joachim Scholz with kind permission). (E) White fungal mycelia are attached to a red me-dieval glass surface (bar = 1.5 mm). (F) Crater-shaped lesions within the gel layer of a me-dieval glass surface are caused by black fungal colonies visible in the lesions (bar = 380 l m).

100 K. Sterflinger

TABLE 1 Most-abundant fungal species on rock (sandstone, marble, and granite), theiracid production and pigmentation

Fungal species in humidclimatea Acidsa,b,c, f Pigmentationb,d

Alternaria alternata tenuazonic melanin, brownAphanocladium album acetic, fumaric, glyoxylic, white

2-oxoglutaric, oxalicAspergillus fumigatus acetic, formic, fumaric, green

gluconic, glyoxylic,itaconic, oxalic

Aureobasidium pullulans formic, fumaric, gluconic, melanin, brownoxalic, oxaloacetic,2-oxogluconic

Botrytis cinerea citric, formic, fumaric, white, grayish-gluconic, lactic, oxalic, browntartaric

Cladosporium cladosporioides none melanin, light todark brown

Cladosporium oxysporum n.d. gray, brownCladosporium sphaerospermum none melanin, brownCladosporium tenuissimum formic, fumaric, gluconic, melanin, brown

lacticConiothyrium cerealis n.d. olive-greenEngydodontium album none whiteEpicoccum purpurascens n.d. red, yellow, brown,

a -carotene,c -carotene,torularhodine,rhodoxanthine

Exophiala jeanselmei melanin, blackFusarium solani acetic, citric, fumaric, solanione, green,

glyoxylic, oxalic bluish-brownHormonema sp. acetic, formic, fumaric, melanin,

glyocylic, tartaric brownish-blackLecythophora sp. oxalic, acetic, citric, fumaric, orange-pink

glyoxylic, gluconicPaecilomyces farinosus citric, fumaric, oxalic white, yellowPaecilomyces lilacinus none vinaceusPaecilomyces variotii citric, gluconic olivePenicillium aurantiogriseum citric, gluconic, fumaric, bluish-green

malonic, glyoxylic, lacticPenicillium brevicompactum citric, fumaric, gluconic, gray-green,

oxalic yellow-greenPenicillium chrysogenum oxalic, citric, gluconic, yellow, green,

malonic, greenish-bluePenicillium citrinum acetic, formic, fumaric, blue-green, yellow

gluconic, glyoxylic,oxalic, kojic, tartaric

Penicillium expansum citric, formic, fumaric, yellow, blue-greengluconic, 2-oxogluconic

Penicillium glabrum citric, formic, fumaric green-gray


TABLE 1 Most-abundant fungal species on rock (sandstone, marble, and granite), theiracid production and pigmentation (Continued)

Fungal species in humidclimatea Acidsa,b,c, f Pigmentationb,d

Penicillium nigricans fumaric, gluconic, gray, olive-gray,glyoxylic, lactic, oxalic yellow, orange

Penicillium purpurogenum acetic, formic, fumaric, green, dark red,glyoxylic, tartaric, kojic reddish purple,

purpurogenone,desoxypurpurogenon,purpuride

Penicillium spinulosum acetic, citric, formic, whitefumaric, glyoxylic,orsellinic, oxalic,tartaric

Phaeococcomyces catenatus n.d. melanin, blackPhaeotheca-like none melanin, brownPhoma levellei none gray-olive, brown,

partly melaninPhoma putaminum nonePhoma terricola noneRhodotorula minuta n.d. torularhodineTrichoderma harzianum acetic, citric, formic, oxalic greenTrichoderma koningii citric, formic, fumaric, green

gluconic, glyoxylic,oxalic

Verticillium lecanii citric, fumaric, gluconic, white, pale yellowglyoxylic, oxalic

Verticillium psalliotae formic, glyoxylic, oxalic, white, red, purple,tartaric brown, oosporein

Fungal species in arid andsemiarid climate c,d ,e, f,g Acidsa,b,c, f Pigmentationb,d

Alternaria alternata n.d. melanin, brownCladosporium sphaerospermum none melanin, brownConiosporium apollinis none melanin, blackConiosporium perforans none melanin, blackConiosporium uncinatum n.d. melanin, blackHormonema dematioides acetic, formic, fumaric, melanin, black,

glyoxylic, tartaric brownHortaea werneckii none melanin, blackLichenothelia sp. n.d. melanin, brown,

blackPhaeotheca sp. n.d. melanin, blackPhoma glomerata n.d. melanin, brownSarcinomyces crustaceus none melanin, blackSarcinomyces petricola none melanin, blackTrimmatostroma sp. n.d. melanin, blackUlocladium chartarum n.d. melanin, brown

Sources: aBraams 1992; bDomsch et al. 1993; cWollenzien et al. 1995; dDiakumaku 1996;eSterflinger 1995; f Gorbushina et al. 1993; gde Leo et al. 1999.

102 K. Sterflinger

sandstone, granite, and other rock types. The taxonomy and terminology for this group offungi provide severe identi� cation problems for mycologists, as discussed in the sectionon phylogeny and taxonomy. Typical habitats of this fungal group are sun-exposed rocksurfaces in hot deserts (Staley et al. 1981), rock surfaces in cold deserts (Onofri et al.1999; Onofri and Friedmann 1999), and semiarid environments (Gorbushina et al. 1993)(Figures 1C and 3A).

The possible relation between the growth of microcolonial fungi and the occurrenceof crater-shaped lesions—pits—on marble surfaces was � rst mentioned by Krumbein et al.(1991). Sterflinger andKrumbein (1997)developed a model of mechanical attackof fungi onrocks, which is described in the section on mechanical forces. In contrast to fast-growinghyphomycetes, which dissolve carbon rocks by acid excretion, the microcolonial fungihave never been shown to produce organic acids in the laboratory (Wollenzien et al. 1995).However, analysis of rock samples by scanning electron microscopy sometimes showed thefungal colonies or single cells in direct connection with etching patterns on otherwise intactcrystals—an indication that acid attack cannot be completely excluded. For this reason, anattack by release of carbon dioxide must be taken into account as one additional factor ofrock attack by fungi.

Black fungi have also been reported to be secondary inhabitants of thick biogeniccalcium oxalate crusts on granite. In these cases, the crusts were generated by bacteria andlater inhabited, penetrated, and partly destroyed by the fungi (Blazquez et al. 1997).

A less studied area of fungal action is the influence of fungi on carbonate precipitation.Nonetheless, a few case studies demonstrate the indirect influence of fungi on carbonateprecipitation. Fungi infect corals in coral-reef formations by penetrating the carbonatematrix. As a defense reaction, the animals start an intensive carbonate nucleation at the pointsof fungal penetration, which results in the formation of carbonate domes on the exoskeleton.The corals also grow upward to escape the fungal invasion (Le Campion-Alsumard et al.1995). A similar phenomenon has been reported for the formation of multilayered bryozoanreefs of Celleporaria sp. collected from Chatham Island, New Zealand (Figure 1D). Fungalattack in this case triggers the self-overgrowth of the bryozoan, which results in multilayeredstructures composed of the bryozoan skeleton and fungal mats. The structures are calledbryostromatolites (Sterflinger and Scholz 1997). Whereas for the corals the capacity ofthe fungi to bore through the carbonate skeleton was described, in the bryozoans the fungientered the zooids through the soft frontal membrane to invade the coelom and degrade thesoft tissue. An attack on the calci� ed exoskeleton was not observed.

Fungal excretion of oxalic acid considerably influences diagenesis of Paleozoic cal-cretes. Calcium oxalate is formed and secondary calcite is precipitated on the pore wallsand thus hardens the micritic matrix around pores. This was very impressively demon-strated by Verrecchia (1990) and Verrecchia and Dumont (1996), who found that fungalhyphae are the most abundant organic features in weathered pro� les of chalky limestone.Oxalate excreted by the fungi dissolves calcium carbonate of limestone and reacts with freeCa2+ , leading to weddelite precipitation. Free carbonate is released, which can contributeto calcium carbonate precipitation outside the pores.

Calcium oxalate precipitation on fungal mycelia is observed through all taxa of thefungal kingdom, including the Zygomycotina (Urbanus et al. 1986) and the Basidiomycotina(Whitney and Arnott 1987). The fungal precipitation of calcium oxalate is considered tobe a major sink of otherwise toxic amounts of calcium in soil and other environments(Whitney and Arnott 1988). Those authors demonstrated that the growth and form of calciumoxalate crystals on hyphae of Gilbertella persicaria (mucorales) and of Agaricus bisporus(basidiomycetales ) are determined by the fungus and by the carbon source available for


fungal growth. Interestingly, Manoli et al. (1997) found that chitin, the major compound ofthe fungal cell walls, favors the deposition of calcite crystals from supersaturated solutionsat pH 8.5 at 25±C.

Dissolution and Precipitation of Metal Ions

In contrast to bacteria, fungi have not been shown to use minerals as an electron sourceto yield energy. Nevertheless, they need trace elements for their metabolism and are ableto change the oxidation state of metal ions and transport the ions into the cytoplasm.Siderophores such as ferrichrome, coprogen, and fusigen are Fe3+ -complexing agents thatenable iron uptake into the cells. Some fungi, for example, Saccharomyces cerevisiae, donot produce siderophores but reduce iron outside the cells before taking up the divalentcation (Cooke and Whipps 1993). Thus the mechanisms to solubilize iron are commonand essential requisites of the fungal metabolism. However, iron and other minerals bearingheavy metalsare also dissolved even if there is no direct need for incorporation of the elementin question into the metabolism. Groudev and Groudeva (1986) studied the dissolution ofiron from quartz-sand by species of Aspergillus and Penicillium. In general, oxalic, citric,and gluconic acid, which are excreted by numerous hyphomycetes (Braams 1992), arestrong solubilizing agents of feldspar, biotite, and phyllosilicates, the latter being the claycement in granite and sandstone (de la Torre et al. 1993) (see Table 2).

The precipitation of iron and manganese as a consequence of fungal creation of anoxidative environment is widespread among fungi (Ehrlich 1981; Schweisfurth 1971, 1972;Schweisfurth and Hehn 1972). However, the use of these reactions for the fungal metabolismis still under consideration. Krumbein and Jens (1981) concluded from � eld analysis thatthere is a close relation between fungal hyphae in rock varnish and manganese deposits onthe hyphae. Grote (1986) showed in laboratory experiments that Alternaria, Cladosporium,Fusarium, Penicillium, and Pleospora strains isolated from rock varnish are able to oxidizeiron and manganese.

Degradation of Silicates

Aluminosilicates and silicates are easily degraded by fungi. The most important mecha-nisms are the production of organic and inorganic acids, alkalis, and complexing agents(Rossi and Ehrlich 1990). CO2 production by respiration affects the weathering of silicatemineral by interacting with the mineral surface. The weathering rate is influenced by theamount of organic matter degraded by fungi and bacteria in the soils. The ratio betweenCO2 released by fungi and CO2 released by bacteria has never been measured in a naturalmixed community. Apparently, whether fungi or bacteria are the more active organismsdepends on the kind of organic matter that is degraded in the soil. Other inorganic acidsattacking silicates, e.g., nitric acid and sulfuric acid, are produced exclusively by bacteria.However, fungi are more active in the production of organic acids. Especially oxalate, astrong solubilizing agent of silicate minerals, is mainly produced by fungi. Among themost important species attacking silicates is Aspergillus niger, which degrades olivine,dunite, serpentine, muscovite, feldspar, spodumene, kalin, and nephelin. Penicillium sim-plicissimum causes degradation of basalt (Mehta et al. 1979). Penicillium expansum andScopulariopsis brevicaulis also release aluminum from aluminum silicates (Rossi 1979).For this reason the molds have attracted attention as commercially (industrially) used bi-oleaching organisms. The attack on glass will be described in the section on weathering ofmonuments.

104 K. Sterflinger

TABLE 2 Fungal species able to accumulate and to solubilize minerals and metals

Fungal species Special features Literature

Alternaria alternata reduction of Fe(III), reduction Grote 1986of Mn(IV)

Alternaria tenuis oxidation of Fe(II) Grote 1986Amanita muscaria adsorption of Pb Vodnik et al. 1998Aspergillus f lavus sul� de oxidation and Wainwright and

accumulation Grayston 1989Aspergillus niger adsorption of Zn, Th, Cd, Cu, Luef et al. 1991;

U; sul� de oxidation and Wainwright andaccumulation Grayston 1989;

Wainwright 1993;Gadd 1990

Aspergillus terreus adsorption of U, Th Gadd 1990Aureobasidium pullulans adsorption of Cu Wainwright 1993Brettanomyces lambicus adsorption of Zn, Cu, Pb Wenzl et al. 1990Candida albicans adsorption of U Gadd 1990Candida robusta adsorption of U Gadd 1990Candida utilis adsorption of Cd, Cu Gadd 1990Chaetomium globosum adsorption of U Gadd 1990Cladosporium cladosporioides oxidation of Fe(II) Grote 1986Cladosporium resinae adsorption of Cu2+ , Wainwright 1993;

corrosion of Al Pope et al. 1989Cladosporium sphaerospermum reduction of Fe(III), oxidation Grote 1986

of Fe(II), reductionof Mn(IV)

Claviceps paspali adsorption of Zn Luef et al. 1991Engydodontium album oxidation of Mn(II) Grote 1986Fusarium solani sul� de oxidation and Wainwright and

accumulation Grayston 1989Gliocladium roseum oxidation of Mn(II) Grote 1986Hansenula anomala adsorption of U Gadd 1990Laccaria laccata adsorption of Pb Vodnik et al. 1998Mucor f lavus adsorption of elemental sulfur Wainwright 1993Mucor racemosus adsorption of Cd Gadd 1990Paxillus involutus adsorption of Pb Vodnik et al. 1998Penicillium brevicompactum oxidation of Fe(II), oxidation Grote 1986

of Mn(II)Penicillium crysogenum adsorption of Zn, Cd, U, Th Luef et al. 1991;

Gadd 1990Penicillium italicum adsorption of Cu, Zn Gadd 1990Penicillium janthinellum adsorption of U Gadd 1990Penicillium notatum adsorption of Al, Cd, Cu, Pb, Gadd 1990

Sn, ZnPenicillium purpurogenum solubilization of rock phosphate Nahas 1996Penicillium rugulosum solubilization of rock phosphate Reyes et al. 1999a, bPenicillium spinulosum adsorption of Cd, Cu, Zn Gadd 1990Pisolithus tinctorius adsorption of lead Vodnik et al. 1998Pleospora infectoria reduction of Fe(III) Grote 1986


TABLE 2 Fungal species able to accumulate and to solubilize minerals and metals(Continued)

Fungal species Special features Literature

Rhizopus arrhizus adsorption of Ag, Cd, Cr, Wainwright 1993;Cu, Hg, Mn, Pb, U, Th Gadd 1990

Rhizopus oryzae adsorption of U Gadd 1990Rhodotorula mucilaginosa adsorption of Cd Gadd 1990Rhodotorula glutinis adsorption of U Gadd 1990Rhodotorula rubra increasing leaching activity of Fournier et al. 1998

Thiobacillus ferrooxidansSaccharomyces cerevisiae adsorption of Cd, U Gadd 1990Suillus bovinus adsorption of Pb Vodnik et al. 1998Trichoderma sp. sul� de oxidation and Wainwright and

accumulation Grayston 1989Trichoderma viride adsorption of Cd, Cu, U Gadd 1990Ulocladium chartarum oxidation of Fe(II), reduction Grote 1986

of Mn(IV)Ulocladium chlamydosporum oxidation of Mn(II) Grote 1986

Sulfur and Phosphate

Soil fungi such as Trichoderma, Aspergillus, Mucor, and Fusarium and some yeasts are ableto oxidize sulfur, dimethylsul� de, dimethylsulfoxide, and thiosulfate and release sulfate tothe environment (Table 2). The polythionate pathway is considered to be the means bywhich fungi oxidize sulfur. Sulfur oxidation from metal sul� des additionally leads to freemetal ions in the surrounding environment, this can be coupled to toxi� cation of the soiland therefore to increased pressure on other organisms that are less tolerant of heavy metals(Wainwright and Grayston 1989). Like Thiobacillus sp., the fungi also � rmly adhere to metalsul� de grains as a prerequisite for metal solubility. Although fungal oxidation rates of metalsul� des are far below the rates of Thiobacillus ferrooxidans and related bacterial species,fungal metal sulfate oxidation is considered to play an important role in soils. Recent studieshave shown that in the natural environment the acidophilic yeast Rhodutorula rubra oftenoccurs in intimate community with Thiobacillus ferrooxidans (Lopez-Archilla et al. 1995).Moreover, Fournier et al. (1998) found that R. rubra has a stimulating effect on growth aswell as on iron sulfate oxidation of T. ferrooxidans. This phenomenon was explained byvitamins and cofactors being excreted by the yeast and favoring growth of the bacterium.

Like some heterotrophic bacteria, fungi also are able to oxidize sulfur to form SO42 ¡ ,

e.g., Fusarium solani and Penicillium luteum. The rates of oxidation are far below thoseof Thiobacilli and other chemolithotrophic bacteria but are assumed to play a role in soils(Wainwright and Killham 1980). Cu, Pb, and Zn sul� des are leached to soluble sulfate byAspergillus niger (Wainwright and Grayston 1986).

Fungi have a greater potential than bacteria to dissolve rock phosphate because thesolubility of the phosphate depends on the decrease in pH and the chelating activity of theorganic acids with minerals containing Fe3+ , Ca2+ , and Al3+ . For example, Aspergillusochraceus and Penicillium implicatum have a very high dissolution capacity for rock phos-phate (Nahas 1996). Two different possible mechanisms for dissolving rock-phosphatefrom hydroxy apatite, iron phosphate, and aluminum phosphate have been described forPenicillium rugulosum. The production of citric or gluconic acid and the extrusion of H+

106 K. Sterflinger

result from membrane transport mechanisms (Reyes et al. 1999a). These processes areinfluenced by the sources of the nitrogen, phosphate, and carbon. Citric acid productionand the resulting amount of phosphate dissolution are increased if nitrate is the only nitrogensource. Because citric acid is involved not only in the dissolution of phosphate but also indissolution of iron and other metals from minerals, the process of nitrate accumulation insoils might play an important role for the weathering of rock in general. What type of acidis produced depends more on the carbon source. With glucose, only citric acid is producedwhereas with sucrose, citric and gluconic acid are produced (Reyes et al. 1999b). In gen-eral, dissolution of phosphate in soil is a very important process for plant growth. Severalstudies have shown that the phosphate uptake by plants can be markedly increased by eithermycorrhizal fungi (Azcon-Aguilar et al. 1986) or inoculation of soil with species capableof solubilizing free phosphate, such as P. bilaii (Cunningham and Kuiack 1992).

Fungi as Biosorbents for Metal Accumulation

Hyphomycetes and yeasts are highly effective biosorbents for various cations: Fe, Ni, Zn,Ag, Cu, La, Pb, Cr, and Mo (Gadd 1990) (Table 2). One kilogram of fungal biomass report-edly can adsorb 63 mg of Cu (Siegel et al. 1990), and as much as 1 g of Zn was adsorbed by1 g of mycelium (Luef et al. 1991). Two different main mechanisms are distinguished for themetal binding by fungi: the metabolism-independent binding of ions to cell walls and otherexternal surfaces as (e.g.) extracellular polymeric substances, and the energy-dependent ac-tive uptake through the cytoplasm membrane. The latter mechanism is carried out by livingcells exclusively, whereas the � rst is a passive process of living and dead fungal biomass.Several factors influence the metal binding capacity of fungi: (1) The radius of the ions(Tobin et al. 1984); (2) the pH of the surrounding environment, with a low pH generallysupposed to decrease the binding capacity (de Rome and Gadd 1987); (3) the biomassconcentration of the fungus, with lower cell densities allowing a higher yield per unit ofbiomass [for Saccharomyces cerevisiae and Aspergillus niger a decrease in the speci� cuptake of ions accompanied an increase of biomass (Gadd et al. 1988)]; (4) the presence ofmelanin, which reportedly plays a major role in the capacity of fungi as biosorbents. Gaddand Mowll (1995) reported that the dark-pigmented and thick-walled clamydospore-likecells of A. pullulans adsorbed three times more copper than less-pigmented yeast-like cellsand mycelium. However, no further investigations have been reported on this phenomenon,which could be caused either by to the presence of melanin or by the fact that the melanizedwalls are usually thick and multilayered and therefore have a better binding capacity thanthinner cell walls. A schematic presentation of major fungal polysaccharides involved inbiosorption is shown in Figure 2. Therefore, the adsorption of mineral compounds mightbe important not only in the soil but especially on surfaces typically inhabited by melanin-pigmented fungi, such as rock. The metal-binding capacity of fungi is important not onlyin industrial applications but also in the natural environment. Vodnik et al. (1998) reportedthat ectomycorrhizial fungi such as Lactarius piperatus, Pisolithus tinctorius, and Suillusbovinus adsorb lead in soil and thus protect plants from taking up toxic amounts of themetal.

Fungal Impact on Fossil Organic Carbon

Coal is one of the most important energy sources on earth, but its transformation into aliquid and clean fuel is an energy-intensive process if carried out thermochemically. Forthis reason, the microbial transformation of coal has attracted much attention within thepast decades. Various studies have convincingly shown that fungi are able to transform coal


FIGURE 2 Schematic presentation of the fungal cell wall components involved in thesorption of metals; melanin is not indicated (Source: Siegel et al. 1990).

into a powdery mass and also to liquefy it (Cohen and Gabriele 1982; Bublitz et al. 1994)(Table 3). Recently, a special issue of Applied Microbiology and Biotechnology (Vol. 52,No. 1, July 1999) was published containing all contributions from the Ninth InternationalConference on Coal Science.

Although the reports on coal-dissolving agents are partly contradictory, one can de-termine from the literature the importance of distinguishing between the highly oxidizedhard coal and low-rank coal with a high yield of lignin compounds that can be degraded bylignin-degrading white rot fungi such as Trametes sp. and Phanerochaete sp. Extracellu-lar oxidative enzymes such as laccase and Mn-peroxidase (Willmann and Fakoussa 1997)and the excretion of ammonium oxalate monohydrate by Trametes versicolor (Cohen et al.1990) play a major role in the degradation of coal. Torzilli and Isbister (1994) found evi-dence that metal chelation by oxalate plays an important role in the coal solubilization byT. versicolor and P. chrysosporium but not by Aspergillus. Hofrichter et al. (1997) reportedthat the mechanical action of fungal mycelia also alters the structure of hard coal by splittingoff small particles. Apparently, for liquefaction and degradation of coal, fungi are superiorto bacteria in the ability to degrade polycyclic lignin compounds. The mechanisms involvedin the process are manifold and include a direct enzymatic, mechanical, and chemical attackof excreted metabolites.

108 K. Sterflinger

TABLE 3 Fungal species involved in bioconversion of coal

Fungal species Special feature Literature

Aspergillus sp. solubilization of low-rank coal Torzilli and Isbister 1994Candida sp. solubilization of coal Quigley et al. 1989Coprinus sclerotigenis liberation of 2-hydroxybiphenyl, Hofrichter et al. 1997

benzenes, and polycyclicaromatic hydrocarbons fromhard coal

Cunninghamella sp. solubilization of coal Quigley et al. 1989Fusarium oxysporum solubilization of coal Holker et al. 1995Paecilomyces sp. solubilization of coal Faison and Lewis 1989Panus tigrius molecular weight loss Hofrichter et al. 1997

of asphaltenePenicillium citrinum solubilization of coal Polman and Stoner 1994Phanerochaete solubilization of low-rank coal Torzilli and Isbister 1994;

chrysosporium Willmann and Fakoussa1997

Trametes versicolora solubilization of low-rank coal Torzilli and Isbister 1994;Willmann and Fakoussa1997

Trichoderma atroviride solubilization of coal Faison and Lewis 1989

aThree synonyms of the species are used in the literature: Polyporus versicolor, Polystictus ver-sicolor, and Coriolus versicolor, of which the � rst, together with Trametes versicolor, is the mostcommonly used.

The same holds true for the degradation of lignin and chlorinated lignin compounds(Bergbauer et al. 1991). The role of fungi in the anaerobic biodegradation and alterationof plant material to form fossil organic carbon still seems to be underestimated. Studies byEkwenichi et al. (1990) on degradation of lignocellulose in elephant grass by Penicilliumand Curvularia species show that this fungal ability plays an important role also in theformation of methane and propane.

Fungi are involved not only in the formation of gases but also in their degradation inthe environment. Fungi extract volatile substances from the atmosphere, e.g., kerosene anddiesel oil, but also extract styrene from waste gases and thus have a potential for use asnatural air-� lter cleaners (Cox 1995). Especially black yeasts are adapted to live on suchcarbon sources: Sterflinger et al. (1999a) showed that Hortaea werneckii, a black yeastfrom hypersaline environments, e.g., salt pans and rocks in sea spray areas, is able to liveon diesel oil aerosol. Cox (1995) presented Exophiala jeanselmei as a possible organismfor removing styrene from waste gases. No studies yet exist that quantify the amount ofhydrocarbons extracted from the air in urban areas by the fungal or bacterial microflorainhabiting the surfaces of buildings, although this process was suggested by Krumbein yearsago (1966).

Fossilized Fungi

The � rst fossil fungal structures were found in the early Devonian Rhynie Chert(McMenamin and McMenamin 1994), in which Taylor et al. (1999) have since discov-ered perfectly preserved perithecia and asci of pyrenomycetes. Fossilized fungi are found aslichenmycobionts in stromatolites(Klappa 1979). Iron stromatolites remarkably resembling


the banded iron formations but nearly exclusively generated by fungal mycelia were � rstreported from the Rhenish Massif near Warstein (Germany) by Kretzschmar (1982). Da-hanayake and Krumbein (1985) detected fossilized branched hyphae and conidiophore-likestructures in Djebel-Onk phosphorites of the Algeria–Tunisia frontier. Taylor and White(1989) interpreted structures in Triassic silici� ed rock as fungal structures of the ichno-taxon Endogonaceae but were not able to � nd a recent equivalent taxon. Also oolithesand oncolites can be generated in fungal mats, as has been demonstrated for Djebel-Onkphosphorites at the Algeria–Tunisia frontier (Dahanayake and Krumbein 1985; Dahanayakeet al. 1985).

Latz and Kremer (1996) reported on fungal mycelia as crystallization nuclei for theformation of rock crystals formed between Oligocene and Miocene. The fungal hyphaewere encrusted by goethite during their metabolic activity and later served as crystallizationnuclei for dissolved silicate. In this crystalline matrix, fungal morphology was so perfectlypreserved that the organisms could be assigned to the recent genera Chaetomium, Preussia,and Papilaspora and various species of the mucorales.

Fungi thus contribute to fossil formation; recent works even hypothesize that the “gar-den of Ediacara” ( « 600 million years ago) might rather be the “garden of Lichens” becausethe special taphonomy of the so-called ichnotaxon Vendobiont might be caused by fossilizedlichen thalli rather than soft body animals (Retallack 1994).

Mechanical Forces

Not only the manifold physiological activities of fungi influence geological processes butalso their morphology and way of growth, which enables them to influence processes ofrock degradation, soil diagenesis, and sediment structures. Because of their ability to formlarge three-dimensional networks in materials, they bind and aggregate particles. Fungicontribute to the stability of soil and sand dunes in coastal marine environments (Forsterand Nicolson 1981). Fungi ramify throughout the soil aggregates and radiate from a centerto bind sand grains together. Because fungi belong to the � rst colonizers of sand dunes,they play an important role in the primary solidi� cation of young dunes that are not yetcolonized by plants.

Given their particle-binding activity in soils, the presence of fungi increases the waterretention of the soils and thus the time for more chemical weathering reactions. More-over, the physical disruption of particles in soils and rocks increases the area of the re-active surface and exposes fresh surface material, thus increasing chemical weatheringprocesses (Barker et al. 1997). Hofrichter et al. (1997), for example, reported that the me-chanical action of fungal mycelia alters the structure of hard coal by splitting off smallparticles.

The mechanical action of fungal growth affects various building materials such as brickand concrete (Gravesen et al. 1994) as well as natural rock. Sterflinger (1995) developed thefollowing model for fungal attack on marble, based on analysis of � eld samples (Figure 3)and long-term experiments using microcolonial fungi inoculated onto Carrara marble: Inthe prepenetration stage, the fungi adhere � rmly to the rock surface by means of appressoria.Then, thin penetration hyphae extend into the inner part of the rock through discontinu-ities between the crystals (Figure 3Ci, E), i.e., the crystallographically determined zonesof weakness. The penetration hyphae follow � ssures and cavities of the rock into internalhollows where new fungal colonies develop (Figure 3Cii). The resulting changes in internalpressure loosen the crystals, which � nally fall down (Figure 3Ciii). The process of penetra-tion, decohesion, and � nal loss of material is progressive because the fungi penetrate deeperinto the rock and a continuous cycle occurs which is assumed to be the cause of biopits

110 K. Sterflinger

FIGURE 3 (A) Natural marble outcrop in the Mediterranean. The dark zones on the rockare crusts of black fungi. (B) Hortaea werneckii is a typical example of black yeasts.The pure culture shown was isolated from the rock in A (bar = 60 l m). (C) Schematicpresentation of fungal attack on crystalline rock: i) penetration stage; ii) growth of rock-internal colonies and loosening of crystals; iii) new exposed surface and continuation ofpenetration. (D) Colonies of meristematic fungi in a pit forming satellite colonies (bar =600 l m). (E) Petrographic thin section of marble showing growth of black fungi betweenthe crystals (bar = 19 l m).

as large as 2 cm in diameter (Sterflinger and Krumbein 1997). Currently, no methods areavailable to observe these relatively slow mechanisms directly or to quantify the internalpressure caused by the growing colonies (Wiggins 1990). Interestingly, melanin has beenimplicated as playing a major role in the abilityof fungi to penetrate hard substances becausethe development of penetration pegs from appressoria depends on the presence of melaninin the cells (Wheeler and Bell 1988). However, for rock-dwelling fungi, Diakumaku (1996)showed that the capacity to dwell in marble was very little or not at all influenced by theinhibition of melanin synthesis with tricyclazol nor was the effect of a biocidal treatmentincreased by the addition of tricyclazol.

The growth of black fungi on white rock surfaces (Figure 3A, B) causes a selectiveabsorption of solar radiation, which leads to a local extension of crystals and thus to dis-continuities in the fabric—which also causes crystal decohesion (Dornieden et al. 1997).


Subsurface Fungi

Within the framework of the Department of Energy’s Subsurface Science Program begunin the 1980s, fungi were isolated from various subsurface environments, e.g., groundwa-ter, rock, and subsurface sediments (Frederickson and Onstott 1996). However, Madsenand Ghiorse (1993) stated that the abundance of fungi in groundwater systems is muchlower than that of bacteria—in part, perhaps, because subsurface life is limited by tem-perature (Fredrickson and Onstott 1996). Given that the highest growth temperature forfungi—D. gallopava at 65±C—is far below that of some thermophilic and hyperther-mophilic bacteria—up to 110±C, one can assume that fungal subsurface life does not reachthe same depth as bacterial subsurface life. Nevertheless, in view of the high resistance ofsome fungi, especially the meristematic fungi and black yeasts, against such stress factorsas oligotrophic conditions and desiccation, and in view of their ability to grow in a widepH range, it seems rather astonishing that only « 100 fungal “types of fungi” were isolatedwithin the framework of the program and at various sampling sites (Fredrickson and Onstott1996). Therefore, one might suspect that the isolation techniques applied by the authorscited above were probably not suitable to isolate the slow-growing black fungi typical forthe lithosphere. These fungi require special isolation techniques, which will be describednear the end of this review.

Weathering of Monuments and Buildings by Fungal Activity

The alteration processes described above occur not only in the natural environment butalso on the material from which buildings and works of art are made. Although in thenatural environment, these processes are necessary for geobiocycling of the elements, theyare undesirable with respect to alteration and destruction of historical buildings. Becausemost of the mechanisms of destruction are the same as described earlier in the section onfungal attacks on minerals, here just a short summary of phenomena is given; the aspect ofaesthetic changes caused by fungi is, however, discussed in detail.

Buildings and works of art are affected by both the mechanical and the physiologicalproperties of fungi. The “sugaring” of rock can result from bacteria and fungi dissolvingthe binding matrix by acid production or by mechanical boring and growth in the rockinterior. Pitting of marble and limestone with cavities as large as 500 l m is caused bythe expansion of lichen fruiting bodies. Black fungi can cause pits as large as 2 cm indiameter, as described earlier. Fungi also have an important impact on the deterioration ofnatural and manmade antique and medieval glass (Krumbein et al. 1991) (Figure 1E, F).Because fungal mycelia adsorb water and keep it on the glass surface, the process of silicahydration and silica gel formation is increased. The mobilization and oxidation of Fe(II)and Mn(II) leads to formation of dark patinas on glass surfaces. Bacteria and fungi producechelating agents that mask the ions dissolved from the glass by chemical processes (e.g.,Ca2+ ). Accordingly the chemical equilibrium is changed and the process of ion dissolutionis increased (Eckhardt 1985). Fissures and cracks are mechanically penetrated by variousorganisms, which causes material loss and enlarges the reactive surface (Krumbein et al.1998).

The optical appearance of natural rock surfaces and building stone differs widely fromfreshly broken rock. Usually, surfaces get darker, witha brownish-green or black appearance,but frequently an orange and green stain of rock is also observed. Depending on climateand exposure, the color change of freshly broken rock is the result of several factors,such as deposition of fly ash and dirt particles, leaching and super� cial deposition ofiron or manganese, development of algal and cyanobacterial bio� lms, and the presence ofpigmented bacteria. Krumbein (1993) and Urzi et al. (1991) give de� nitions of the term

112 K. Sterflinger

crust and patina and demonstrate in several examples how biogenic pigments (includingcarotenoids, melanin, and chlorophyll) influence the optical appearance of rock monuments.Krumbein (1993), moreover, interpreted the color change of the Acropolis in Athens, whichhas been documented over the last 150 years in a series of paintings, as being a consequenceof climatic changes that have influenced the microbial community on and in the marble ofthe Parthenon. Fungi play a major role in the color change of rock surfaces (Figure 4A, B).

In a combined approach of microbiological and geochemical methods Sterflinger et al.(1999b) demonstrated a direct correlation between orange pigmentation (patination) ofgranite and sandstone and the presence of rock-inhabiting fungi. On the basis of literaturedata, pigments were identi� ed as different carotenoids that were typical for the organismsunder investigation. In addition, by using the fungi isolated from � eld samples, patinas werearti� cially produced on freshly cut marble cubes. Within the frame work of the Eurocare–Euromarble program, marble cubes were exposed in the center of Munich and Vienna in1990. Sterflinger (1999) investigated the fungal microflora of these cubes after 8 years ofexposure and interestingly found Epicoccum purpurascens to be one of the most commoninhabitants of marble exposed to moderate climaticconditions in Northern Europe. Becausethis fungus produces a wide variety of pigments, including b -carotene, c -carotene, toru-larhodine, and rhodoxanthine as well as humic acid-like products and melanins (in conidia),the rocks inhabited by E. purpurascens are stained brownish to yellowish (Figure 4A, B).

Although orange patination can also be produced by pigmented bacteria, the develop-ment of black crusts on rock is mostly attributable to fungi and only partly to cyanobacteria.The black and brown crusts can also be microbially induced manganese and iron precipita-tion on rock where fungi and bacteria are involved.

Ecophysiology of Epi- and Endolithic Meristematic Fungi

Rock surfaces provide very special living conditions for epi- and endolithic organisms.Because of extreme temperatures, rapid changes in the water activity, high-UV radiation,and oligotrophic nutrient conditions, this habitat is often termed an extreme environment(Wainwright 1993). In coastal regions, the rock surfaces are additionally characterized byhigh amounts of salt, and therefore osmotic stress influences what rock microflora is present(Auger 1989).

The rock surface is supposed to be an oligotrophic habitat with respect to organic car-bon. In fact, for fungal growth, organic carbon is probably not the most important problem,being provided from air in the form of dust particles, aerosols (mainly aliphatic and aro-matic hydrocarbon), and partly also fossil organic carbon that is � nely distributed betweenthe mineral compounds of biogenic sedimentary rocks. Fungi are able to use a very diverse

!FIGURE 4 (A) Variety of fungal colonies isolated from rock, showing yellow to brownpigmentation from carotenoids. (B) The orange-brown patina on Schoenbucher sandstonewas generated by Epicoccum nigrum (bar = 625 l m). (C) The detection of fungal myceliaon rock is made possible by calcofluor white staining (bar = 50 l m). (D) A high-precisionnegative replica is made for the nondestructive optical analysis of a marble surface. (E) Flu-orescent in situ hybridization of an epilithic fungus allows taxon-speci� c detection. Theprobe is marked with rhodamine (bar = 75 l m) (photo: Matthias Hain with kind permis-sion). (F) Fingerprints of six strains of epilithic fungi from Alpine regions. The methodallows reliable identi� cation and differentiation of morphologically similar strains. The18S rDNA was digested with the restriction enzyme Rsa I. Note that strains on lanes 1 and2 are identical (m = marker Lambda DNA, Pst I-digested).


114 K. Sterflinger

range of hydrocarbons. Species of Cunninghamella , for example, can use naphthalene, ben-zopyrene, benzoanthracene, and methylcholanthrene. This is especially interesting becauseCunninghamella sp. were also isolated from subsurface environments in the framework ofthe Subsurface Science Program (Balkwill and Sterflinger, unpublished data). Evidence offungi living on volatile substances has also come from investigations of surfaces in man-made environments: hyphomycetes such as Cladosporium, Penicillium, and Acremoniumare inhabitants of microglass air � lters in hospitals and are assumed to absorb organic mate-rials from the air flow (Simmons et al. 1997). In fact, many fungal species, e.g., Aspergillusflavus, A. niger, Fusarium oxysporum, F. solani, Gliocladium viridens, Mucor rouxii, Peni-cillium crysogenum, and others, are able to grow under oligocarbotrophic conditions. Thiswas demonstrated by growth on what is regarded to be practically a nutrient-free medium:silica gel (Parkinson et al. 1989; Barakah 1992). Several studies have provided evidencethat fungi are able to � x CO2 from the atmosphere and incorporate it into cell material.Palmer and Friedmann (1988) tested two strains of cryptoendolithic fungi for the ability toincorporate inorganic carbon and found substantial uptake of CO2 by a darkly pigmentedfungus that they assumed to be a parasymbiont but which today would most probably beidenti� ed as a free-living meristematic fungus. Although real chemolithoheterotrophy hasnot yet been proved for fungi, evidence shows that energy for CO2 � xation can be gainedfrom thiosulfate or from ammonium oxidation (Wainwright 1993).

Meristematic fungi are especially adapted to grow on surfaces characterized by changesbetween two environmental situations: high temperature combined with matrix stress be-cause of low water availability, and moderate temperatures combined with availability ofwater. That the temperature tolerance of rock-inhabiting meristematic fungi increases withincreasing dehydration of the fungal thallus was found for fungi living on rock deserts rocksin Arizona, California, Oregon, Australia, and Egypt by Palmer et al. (1987, 1990) and formeristematic fungi from the Mediterranean in a series of laboratory experiments (Sterflinger1998). Air-dried mycelia of some meristematic fungi tolerate temperatures as hot as 120±Cfor at least 0.5 h (Table 4). As a response to temperature stress, multilayered cell wallsare developed and trehalose is accumulated, whereas under NaCl stress, the intracellular

TABLE 4 Temperature and NaCl tolerance of epilithic black fungi

Max. temperature Max. temperature Max. NaCl conc.Fungal species/strain at 100% RH [±C] at 0% RH [±C] [%]

Capnobotryella-like 35 80 0Coniosporium apollinis 60 105 0Exophiala sp. 45 105 7Sarcinomyces crustaceus 50 80 3.5Hortaea werneckii 45 80 27Coniosporium perforans 55 85 7GU 3 50 90 3.5Sarcinomyces sp. 50 95 0Phaeotheca � ssurella 55 95 14Taeniolella fagina 45 85 14Black yeast 70 120 14Cladosporium 50 85 21

sphaerospermumTrimmatostroma sp. 65 85 10.5

RH, relative humidity (Source: Ster� inger, 1999).


amount of glycerol regulates the osmotic potential. Wollenzien et al. (1995) suggested thatthe endoconidiation observed in such genera as Sarcinomyces and Phaeotheca is an adap-tation to hostile environmental conditions. Sterflinger and Krumbein (1995) showed that acombination of stress factors forces different fungal genera with hyphomycete morphologyto form meristematic colonies, even in culture. Palmer et al. (1990) studied the distributionof fungi and of lichen on granite in Oregon and came to the conclusion that the southern-exposed surfaces are dominated by microcolonial fungi, an indication that these are evenmore tolerant against desiccation, high temperature, and UV radiation than lichens.

The third environmental factor that influences the growth on rock surfaces, at leastin areas near the sea, is osmotic stress. Although some strains show a remarkably hightolerance against NaCl, only Hortaea werneckii isolated in the sea spray area on Delos ishalophilic (Table 4). Other strains of H. werneckii isolated from human skin and from saltpans are reported to be adaptive toward NaCl but without real halophily (Zalar et al. 1999a).

Differences in temperature- and osmotolerance exist between species, but no clear linecan be drawn between black fungi isolated from the Mediterranean and species isolatedfrom North German monuments. Concerning the parameters tested here, stress toleranceseems to be obligate in various species of meristematic fungi.

Phylogeny and Taxonomy of Epi- and Endolithic Meristematic Fungi

As mentioned earlier, the rock-inhabiting fungal flora can be clustered into the group of thewell-characterized molds more or less resembling the soil microflora and the meristematicblack fungi. Within recent years, the latter group has been determined to include typicalrock inhabitants in arid and semiarid areas as well as osmotic environments. Several termshave been applied to these fungi in the literature: (1) Staley et al. (1981) termed themmicrocolonial fungi because of their extremely slow growth. (2) Wollenzien et al. (1995)applied the term meristematic fungi because the fungi form slowly expanding, cauliflower-like colonies and reproduce by isodiametric enlargement with subdividing cells. Liberationof propagules is by disarticulation (sarcinic conidiogenesis) or disruption (endogenousconidiogenesis). Additional blastic conidia may be present in low numbers, often changinginto budding cells.(3)The more general termDematiaceaehas been applied to stress the darkpigmentation of the cell walls that are encrusted with melanin. (4) Fungi that additionallyexhibit yeast-like states are called black yeasts (de Hoog and Hermanides-Nijhof 1977).

Given the insuf� cient rates of differentiation and the morphological plasticity (Minter1987), the identi� cation of meristematic fungi and black yeasts is often quite dif� cult.Until now, the rock-inhabiting fungi have been classi� ed in the following anamorphic gen-era: Exophiala Carmichael (Braams 1992), Sarcinomyces Lindner (Wollenzien et al. 1996),Phaeosclera Sigler(Sigler et al. 1981), Botryomyces de Hoog and Rubio (de Hoog and Rubio1982), Lichenothelia Henssen (Henssen 1987), Trimmatostroma Corda (Sterflinger et al.1999a), Capnobotryella Sugiyama (Titze and de Hoog 1990), Hortaea (Horta) Nishimuraand Miyaji (Sterflinger et al. 1999b), Phaeococcomyces (Sterflinger 1999), and Coniospo-rium Link ex Fries (Sterflinger et al. 1997). Because the teleomorph connections of the fungicannot be clari� ed on the basis of morphologic molecular approaches, including analysis ofnuclear 18S rDNA sequencing, internal transcribed spacer (ITS) sequencing and restrictionfragment length polymorphism data were applied to identify and clarify the phylogeneticrelationships of meristematic fungi (Sterflinger et al. 1997). Although the morphology ofthe rock inhabitants is very similar or alike, their origins are polyphyletic. Phylogenetictrees based on 18S rDNA data show that they belong or have close af� nities to at least threedifferent orders of the fungal kingdom: Chaetothyriales, Dothideales, and Pleosporales. Aphylogenetic tree of ascomycetes is shown in Figure 5.

116 K. Sterflinger

FIGURE 5 Phylogenetic tree of ascomycetes. The tree is based on complete 18S rDNAsequences and calculated with the neighbor-joining method. Bootstrap values (%) are gen-erated from 500 trees. The names of epi- and endolithic meristematic fungi and black yeastsare printed in bold letters.

Methods for Studying Fungi in the Geosphere

The study of geomicrobiology involves both the study of the organisms and the study of thematerials, but � rst and foremost it involves the study of the interactions between organismsand the materials they colonize. For this reason geomicrobiological investigations startwith careful observation and necessitate a trained eye for the phenomena in the natural


environment, from the macroscopical to the microscopical scale. Phenomenology thus isone of the most important and one of the most dif� cult tasks for geomicrobiologists. Tounderstand the biological impact on the alteration of a monument or natural rock, it isimportant to document the physical and chemical properties of the material itself as well asthe localitywith all its environmental factors: temperature, humidity, sun exposure, climaticevents, possible nutrient sources, and so forth. For this reason geomicrobiological studiesare also highly interdisciplinary and bring together the knowledge of biologists, chemists,geologists, and physicists.

Sample collection is followed by microscopical investigations, including the use of lightand electron microscopy. To make the visualization in the light microscope easier, severaldifferent staining techniques can be applied. The periodic acid Schiff staining (Whitlachand Johnson 1974) will stain all the polysaccharides in a sample—including bacteria, ex-tracellular polymeric substances, and fungi—and thus obviates a differentiation betweenorganism group. Calcofluor white can be used for speci� c staining of fungal hyphae, whichwill appear bright blue under UV light (Figure 4C). This type of staining sometimes failsfor meristematic fungi because it does not penetrate into the thickened and melanizedcell walls. Only recently have new methods based on genetic information been applied tovisualize fungi on materials. In situ hybridization with fluorescent marked DNA probes(Figure 4E) allows the detection of speci� c taxa directly on the sample without previousisolation (Sterflinger and Hain 1999). One problem with this method, however, is that thedatabanks containing the DNA sequences necessary for the design of taxon-speci� c probesare still small. Consequently, the sequence of fungal DNA must be determined before themethod can be applied to a broad range of fungi.

Another approach for the detection of fungal species in a natural sample without culti-vation is the use of denaturing gradient gel electrophoresis (Kowalchuk et al. 1997). In thisapproach the total DNA of a natural sample is extracted, the fungal 18S rDNA is ampli� edin the polymerase chain reaction with fungal-speci� c primers, fragments are separated ona denaturing gradient gel, and � nally the sequences of the fragments are determined. Thesequences allow identi� cation. The method was developed in bacteriology and is currentlyadapted to the special requirements of fungi on rock (A. A. Gorbushina, personal communi-cation). Also here the lack of data in the databanks hampers the applicability of the method.However, because DNA sequencing and genetic � ngerprinting are highly important for theidenti� cation of black yeasts and meristematic fungi (Figures 4F and 5), the databanks aresteadily growing and future progress is within sight.

In situ studies of epi- and endolithic fungi are best carried out by scanning electronmicroscopy (SEM) and by petrographical thin sections of samples embedded in epoxyresins, e.g., according to the descriptions of Spurr (1969). Golubic et al. (1975) describeda method for preparing casts of the microboring networks in rock, by which the organismsare � xed within a polymeric resin and the surrounding rock matrix is later dissolved. Thealteration of a surface underlying epilithic organisms can be studied after maceration of theorganisms from the surface with “Eau de Javelle” (Gehrmann et al. 1992).

In cases where sampling is not allowed because of the cultural value of the object,high-precision replicas can be made on the surface. The “negative replicas” (see Figure 4D)are transferred into a “positive replica” in the laboratory, and the positive can be studiedby SEM. The materials and methods used for this method were described in detail bySterflinger and Krumbein (1995). To isolate fungi without sample collection, very smallpieces of material can be picked with thin needles. The pieces are directly transferred togrowth media. However, only a true sampling allows the quantitative (cell counts) analysisof infections with subsequent inoculation and cultivation.

Fungi are usually isolated from water and rock samples by using the diluting platetechnique and relatively rich media, e.g., potato dextrose-, Czapek-, and malt extract agar

118 K. Sterflinger

containing bacteriocidic substances. The isolation and cultivation of rock-inhabiting blackfungi requires a more-selective isolation technique and special media (Wollenzien et al.1995) because the black fungi are slow-growing and will otherwise be overgrown by fast-growing hyphomyetes. Fungal colonies can be picked from the substratum with sterileneedles under a dissecting microscope and can be directly transferred to media. Severalweeks may pass before a fungal colony can be seen with the naked eye, and plates haveto be checked for contaminants daily. A culture medium that can be used for the selectiveenrichment of black fungi contains dichlorane, rosa bengal, and chloramphenicol (Kinget al. 1979); this toxic mixture decreases growth velocity of fast-growing hyphomycetes. Aselective medium for black yeasts containing meso-erythrol as the sole carbon source wasdesigned by de Hoog and Haase (1993).

Because the techniques to study pure and mixed cultures of fungi and their physiologicalabilities in the laboratory are part of the common microbiological and mycological work,they will not be described here in detail; just some special techniques to investigate casesof geomicrobiological importance will be described. If laboratory experiments are carriedout with fungi inoculated on the natural substratum, e.g., rock, the techniques used toinvestigate alteration are as described above. Additionally x-ray tomography can be usedto view changes in the rock fabric during growth of the fungi without destruction of thesample (Diakumaku, 1996).

Mineralizations on the material surface and on the fungal structures can be analyzed byusing a SEM equipped with energy-dispersive x-ray analysis (EDX). Transmission electronmicroscopical analysis combined with EDX can be used for the more detailed localizationofmetal minerals in and on cell walls (Brown et al. 1998). A quantitative analysis of elementsin the sample is possible with atomic absorption spectrophotometry (Luef et al. 1991).

Fungal biopigments such as carotenoids often cannot be optically distinguished fromiron oxide precipitations. Also melanins can resemble manganese–iron precipitations. In thelatter case, infrared spectroscopy can be used for the analysis of melanins in rock samplesand from cultures (Diakumaku et al. 1995); whereas UV–visible spectroscopy and massspectroscopy are suitable for detecting fungal carotenoids extracted from natural rock andfrom pure cultures (Sterflinger et al. 1999b).

Summary

As mentioned in the introduction fungi are often put in the second place by geomicrobi-ologists. However, the data summarized in this review show that fungi not only influencegeological processes by their physiological properties but—and this is a considerable dif-ference from bacteria—also exert some physical forces because of their ability to formhyphae, to penetrate hard substrates, and to spread on and through materials. To get a moredetailed picture of fungal activities, it will be necessary to develop new methods that allowthe speci� c detection of fungal metabolic activity.

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FungiasGeologicAgents - Jagiellonian Universityeko.uj.edu.pl/mycorrhiza/MKBL_2013/konw/10_grzyby jako... · 2013-11-19 · FungiasGeologicAgents 103 fungalgrowth.Interestingly,Manolietal.(1997)foundthatchitin,themajorcompoundof