Geobiology (2007), 5, 265–280 DOI: 10.1111/j.1472-4669.2007.00107.x PhosphorousBlackwellFungalORIGINAL dissolution Publishing ARTICLES of Ltdapatite availability influences the dissolution of apatite by soil fungi A. ROSLING,1 K. B. SUTTLE,1 E. JOHANSSON,2 P. A. W. VAN HEES2 AND J. F. BANFIELD1 1Department of Earth and Planetary Science, University of California, Berkeley, Mc Cone Hall, Berkeley, CA 94720-4767, USA 2Man-Technology-Environment Research Centre, Department of Natural Sciences, Örebro University, SE-701 82 Örebro, Sweden ABSTRACT Apatite (Ca10(PO4)6(OH,F,Cl)2) is the primary inorganic source of phosphorus in the biosphere. Soil fungi are known to increase plant-available phosphorus by promoting dissolution of various phosphate minerals. Yet no apatite dissolution studies exist using fungi as weathering agents, and regulation of fungal weathering activity in response to different levels of phosphorus availability is largely unknown. Fungi were isolated from a grassland soil in northern California. Three pathways of tri-calcium phosphate (Ca3(PO4)2) (TCP) dissolution in liquid culture were identified among biogeochemically active fungi: (1) acidifi- cation (pH 3.3 ± 0.16), (2) moderate acidification (pH 4.9 ± 0.11) and (3) no acidification. Isolates representing pathway 1 and 2 were Zygomycetes in the order of Mucorales. All non-acidifying isolates in pathway 3 were Ascomycetes and cleared the media by altering TCP into hydroxyapatite (Ca10(PO4)6(OH)2) and sequestering it within mycelial spheres. One isolate representing each pathway was used in fluorapatite dissolution experiments either with the fungi present or under abiotic conditions using cell-free liquid media conditioned by fungal growth at different phosphorus and calcium availabilities. Both Mucorales isolates acidify their substrate when growing in the presence of phosphorus. Mucorales exudates were mainly oxalic acid, and conditioned cell-free media with phosphorus induced fluorapatite dissolution at a rate of 10–0.9±0.14 and 10–1.2±0.22 µmol P m–2 s–1. The ascomycete isolate on the other hand, induced fluorapatite dissolution at a rate of 10–1.1±0.05 µmol P m–2 s–1 by lowering the pH of the media under phosphorus-limited conditions, without producing significant amounts of low molecular weight organic acids (LMWOAs). Oxalate strongly etches fluorapatite along channels parallel to [001], forming needle-like features, while exudates from the ascomycete-induced surface rounding. We conclude that while LMWOAs are well-studied weathering agents, these do not appear to be produced by fungi in response to phosphorus-limiting growth conditions. Received 27 October 2006; accepted 20 February 2007 Corresponding author: Anna Rosling. Tel.: +46-18-67 18 64, Fax: +46-18-67 35 99; e-mail: [email protected]. In vitro, phosphorus-solubilizing fungi and bacteria INTRODUCTION produce a clear zone around their growing colony when After nitrogen, phosphorus is the most frequently limiting grown on solid media enriched with a precipitated calcium macronutrient for plants. In soil, free phosphorus is rapidly phosphate. This in vitro solubilization method has been used immobilized by sorption onto minerals or hydrous oxides of for the last hundred years as an initial criterion by which to Al, Fe and Mn. To survive in soil, roots and microbes need identify isolates with the potential to release phosphorus from mechanisms to increase phosphorus availability and facilitate minerals (Whitelaw, 2000). Detection of fungal clearing is uptake (Raghothama, 1999). Many soil fungi are able to facilitated in liquid cultures since mycelial cover does not solubilize phosphorus directly from minerals such as apatite obscure detection of clear zones, but the method is not (Ca10(PO4)6(OH,F,Cl)2). Both plant-symbiotic and free-living suitable for large-scale screening. Microbial solubilization of soil fungi have been demonstrated to increase phosphorus phosphorus in liquid cultures has been demonstrated to uptake in plants through their biogeochemical activity depend linearly on the induced acidification of the growth (Wallander et al., 1997; Whitelaw, 2000). substrate (Gupta et al., 1994). This acidification results from © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd 265 266 A. ROSLING et al. microbial release of organic acids, proton efflux over the between 3 and 5, organic dissolution rates exceed inorganic plasma membrane, and the formation of carbonic acid in the (Welch et al., 2002). In general, dissolution of apatite in biotic media from respiratory CO2 production (Burford et al., 2003). systems is slower than abiotic dissolution, but accelerates as Apart from lowering the pH, organic acids form strong microbial biomass and organic compound concentrations complexes with cations, thereby increasing phosphorus mineral increase (Welch et al., 2002). Although phosphorus-solubilizing dissolution rates. This makes organic acids more effective activity in fungi is well documented, apatite dissolution exper- weathering agents than inorganic acids at the same pH (Gadd, iments with fungi as weathering agents are missing. 1999). Fungal-induced phosphorus solubilization from phosphate Biochemical, morphological, and physiological responses to minerals appears to depend on the amount and composition phosphorus deficiency in plants have been well studied. In of organic acids produced by the fungi (Whitelaw, 2000). dicots, increased production and excretion of organic acids A substantial body of literature exists on the production of and chelators can increase P availability from inorganic organic acids by filamentous fungi, largely because Aspergillus sources. In all plants, the induction of both intracellular and niger is used for the commercial production of citric acid extracellular acid phosphatases production releases P from (Magnusson & Lasure, 2004). When grown under conditions organic sources (see Raghothama, 1999). In yeast and of excess carbon relative to other nutrients (e.g. phosphorus), filamentous fungi, regulation of genes encoding membrane P some fungi maintain a high carbon flux through their mycelia transporters and phosphatases in response to P deficiency has by exuding excess carbon as metabolic intermediates such as also been demonstrated (Oshima, 1999; Tasaki et al., 2004a, b). citric and oxalic acid (Gadd, 1999; Gallmetzer & Burgstaller, Yet, regulation of biogeochemical activity in fungi in relation 2002). Substrate acidification and organic acid production in to phosphorus limitation is poorly understood. Gibson and fungi are strongly affected by the source of nitrogen supplied Mitchell (2004) found no differences in phosphorus solubili- in the growth media. Response patterns to ammonium and zation by four ericoid mycorrhizal fungi grown on different nitrate sources vary in different fungi (Gadd, 1999). This levels of available phosphorus. Gharieb (2000) studied difference can be avoided by providing an organic nitrogen dissolution of gypsum by A. niger and found that dissolution source, for instance L-alanine, to the growing fungi occurred only when phosphorus was supplied to the substrate. (Mahmood et al., 2001). The mycelial growth of ectomycorrhizal fungi has been The biogeochemical significance of organic acid production demonstrated to increase in response to low phosphorus in fungi has been extensively studied with respect to both availability as a result of increased carbon allocation from the bioremediation of toxic metals (Gadd, 1999) and mineral host plant (Ekblad et al., 1995). weathering above and below ground (Burford et al., 2003). This paper examines patterns of apatite dissolution under Apart from substrate acidification and organic acid production, conditions induced by three biogeochemiclly active soil fungi. fungi may induce mineral weathering through nutrient uptake We describe how these fungi regulate weathering in response and physical force (Burford et al., 2003), production of to different phosphorus and calcium availabilities. Rotating extracellular organic polymers mediating processes at the liquid media batch experimental systems were used to measure mineral surface (Barker & Banfield, 1996), siderophore solubilization of phosphorus from apatite. Fungal weathering production (Callot et al., 1987) and cation biosorption to in such systems is limited to substrate acidification and exchange sites in the cell wall (Marschner et al., 1998). Further- activities of fungal exudates, since physical contact between more, the hyphal growth mode of filamentous fungi is well hyphae and minerals is limited. However, by determining adapted for exploitation of nutrient sources in the highly hyphal cation exchange capacities (Marschner et al., 1998) heterogeneous soil environment (Robson, 1999). The ability under different growth conditions, the current study provides of mycelia to connect distant mineral and carbon sources insight into how cell wall composition is regulated in response enables translocation of heterogeneously distributed nutrients to phosphorus-limiting conditions. In soil, fungal weathering and moisture through the mycelia (Hirsch et al., 1995). This may be controlled by processes at the hyphal surface as well as makes fungi potentially important agents of weathering and by the activity of exuded compounds. nutrient movement in the soil system (Finlay & Rosling, 2006). MATERIAL AND METHODS Although apatite is the predominant form of mineral phosphorus in the Earth’s crust, biological forms (i.e. dental Identification of biogeochemically