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Influence of nitrogen source on the solubilization of natural gypsum (CaSO4. 2H2O) and the formation of calcium oxalate by different oxalic and citric acid-producing fungi
Mycol. Res. 103 (4) : 473–481 (1999)
473
Printed in the United Kingdom
Influence of nitrogen source on the solubilization of natural gypsum (CaSO4 . 2H2O) and the formation of calcium oxalate by different oxalic and citric acid-producing fungi
M O H A M M E D M. G H A R I EB1 A N D G E O F F R E Y M. G A D D2* " Department of Botany, Faculty of Science, Menoufia University, Shebein El-Koom, Egypt # Department of Biological Sciences, University of Dundee, Dundee, DD1 4HN, Scotland, U.K.
The ability of six fungi to solubilize natural-occurring gypsum was tested in vitro. The solubilization process was monitored by the production of a clear zone (halo) around or underneath the growing colony on Czapek–Dox agar containing 0n5 % (w\v) gypsum (CaSO ;2H O). Aspergillus niger, Penicillium bilaii, P. simplicissimum and Paxillus involutus displayed differential solubilization activities % # depending on the supplemented nitrogen source. Colonies grown on nitrate-containing medium showed the ability to solubilize gypsum, but when ammonium (at equivalent nitrogen) was used there was a significant reduction in solubilization. It was found that liquid cultures of nitrate-grown fungi produced substantial amounts of oxalic acid, whereas in ammonium-containing medium oxalic acid was only detected in small amounts. The production of citric and gluconic acid under these experimental conditions was low in both media, although the involvement of citric acid in gypsum solubilization is possible. Coriolus versicolor and Phanaerochaete chrysosporium did not exhibit any solubilization activity in nitrate- or ammonium-containing medium. Additionally these two fungi excreted small quantities of oxalic acid in both media with no citric acid being produced in liquid medium. Concomitant with the solubilization process and inside the clear solubilized zone, A. niger, Pax. involutus and P. bilaii produced crystals of differing shapes and abundance depending on the fungal strain. No crystals were produced by P. simplicissimum. These crystals were identified as calcium oxalate based on HPLC analysis and energy-dispersive X-ray microanalysis. Abundance of these crystals was found to be correlated with both oxalic acid production and the acidity of the medium. It is concluded that gypsum solubilization was predominantly achieved by both oxalic acid, which was accompanied by formation of calcium oxalate crystals, and citric acid production rather than the acidity of the medium. The results are discussed in relation to the significance of such an activity in agricultural applications, e.g. land reclamation, as well as the possible roles played by these fungi in mineral cycling.
The process of mineral solubilization by heterotrophic microorganisms has been apparent for many years. Recently, this phenomenon has received attention regarding biotechnological applications for leaching and recovery of metals from low grade ores and biogeochemical cycling of nutrients (Franz, Burgstaller & Schinner, 1991 ; Burgstaller & Schinner, 1993 ; Morley et al., 1996 ; White, Sayer & Gadd, 1997). In agriculture, as a result of the essentiality of phosphorus for plant growth, attention has been paid to the solubilization of insoluble mineral phosphates by soil microorganisms and numerous studies have highlighted the significant role played by soil fungi (Kucey, 1987 ; Asea, Kucey & Stewart, 1988 ; Lapeyrie, Ranger & Vairelles, 1990 ; Jones et al., 1991). A commercial formulation of Penicillium bilaii spores has been registered in Canada to increase available phosphate to wheat (Cunningham & Kuiack, 1992). Different mechanisms of solubilization have been identified including proton excretion, and the production of organic acids, and other chelating metabolites (Agnihotri, 1970 ; Hughes & Poole, 1989 ; Lapeyrie et al., 1990 ; Sayer & Gadd, 1997). Cunningham & Kuiack
* Corresponding author.
(1992) demonstrated that oxalic and citric acid and acidity resulting from ammonium incorporation are significant components of laboratory culture phosphate solubilization by P. bilaii. Oxalic acid is a common metabolite produced by several free-living and symbiotic fungi, and is implicated in the weathering of soil minerals, wood decay, building destruction and solubilization of metal-containing insoluble compounds (Graustein, Cromack & Sollins, 1977 ; Cromack et al., 1979 ; Jones, Wilson & Tait, 1980 ; Malajczuk & Cromack, 1982 ; Jones et al., 1991 ; Douglas & Singh, 1995 ; Sayer & Gadd, 1997). The effectiveness of oxalic acid is attributed to its behaviour as a proton source and the chelating ability of the oxalate anion (Jones et al., 1980 ; Sayer & Gadd, 1997). The most widespread form of naturally-occurring insoluble sulphate is the dihydrated form of calcium sulphate, gypsum (CaSO ;2H O), which occurs in several soils particularly % # those in arid and semi-arid areas (Nelson, 1982). Gypsum is also a by-product of various manufacturing operations and is introduced in building construction. During the last few years the application of gypsum to soil has become attractive because of the resulting improvement in soil properties and the yield of certain crop plants (Alva, Gasho & Guang, 1989 ; Kant & Kumar, 1992 ; Burkert & Marschner, 1992 ; Reading,
Fungal solubilization of gypsum Harrison & Kvien, 1992 ; Ellington, Badawy & Ganning, 1997). Wallace (1994 a, b) reported that gypsum is a key ingredient of many agricultural soils because it can improve physical and chemical properties of soil thus making a better environment for plant growth. In a previous study, we have demonstrated the ability of Aspergillus niger and Serpula himantioides to solubilize gypsum and release sulphate (Gharieb, Sayer & Gadd, 1997). The aim of this study was to investigate the activity of a number of organic acid-producing fungi, particularly citric and oxalic acid, in gypsum solubilization, to characterize this phenomenon further, and to provide details about the probable mechanism(s) involved.
MATERIALS AND METHODS Organisms, media and culture conditions The fungi used were Aspergillus niger Tiegh. (ATCC 201373), Penicillium bilaii Chalab. (kindly obtained from Dr C. Kuiack, Philom Bios Inc., Saskatoon, Canada), P. simplicissimum (Oudem.) Thom (Dr D. Leake, Imperial College, London) Paxillus involutus (Batsch) Fr. (Dr J. Frankland, Institute of Terrestrial Ecology, Merelewood), Phanaerochaete chrysosporium Burds. (Dr N. White, University of Abertay, Dundee) and Coriolus versicolor (L.) Que! l. (Prof C. S. Evans, University of Westminster, London). All were maintained routinely on malt extract agar (MEA, Lab M) at 25 mC. For experiments Czapek–Dox liquid medium was used of the following composition (g l−" distilled water) : sucrose, 30 ; KH PO , 1 ; # % MgSO ;7H O, 0n5 ; KCl, 0n5 ; FeCl , 0n001. Two different % # $ nitrogen sources were used ; either NaNO or NH Cl at $ % concentrations of 2 and 1n27 g l−" respectively. Two 7 mm diam. discs from the margin of 7 d old colonies were used as an inoculum in triplicate cultures of 50 ml liquid medium. 15 g l−" agar (Lab M, No. 2) was used for preparation of solid Czapek–Dox medium. High purity natural gypsum (CaSO ;2H O) was obtained from R. G. Widdowson % # (Scarborough, Yorks, U.K.). The particles were powdered in a ball mill, and the composition and purity verified by powder X-ray diffraction using a Hilton–Brooks X-ray diffractometer fitted with a curved graphite monochromator, using Cu Kα primary radiation. The obtained powder was then passed through a 250 µm sieve. A stock suspension in sterile ddH O # was prepared after dry autoclaving (for 15 min at 121m) of a known weight. To obtain a constant thickness of agar, 10 ml aliquots of agar medium (in triplicate) were autoclaved, and after cooling to 55m and just before pouring, an aliquot volume from the stock gypsum suspension was added to obtain a final concentration of 0n5 % (w\v). Control (gypsum-free) as well as sulphur-free treatments (in which MgCl was used instead of # MgSO ;7H O) were also prepared. Before pouring into % # 90 mm diam. Petri dishes, the media were mixed well to ensure homogeneous distribution of gypsum particles. After setting of the agar and prior to inoculation, " 80 mm diam. discs of autoclaved sterile dialysis membrane (BDH, Poole, U.K.) were placed under aseptic conditions onto the surface of the agar in each Petri dish (Sayer, Raggett & Gadd, 1995). The membrane allowed passage of nutrients\metabolites between the fungus and the agar and provided a convenient means of
474 removing the mycelium. Agar medium was inoculated with 7 mm diam. discs of mycelium cut from the margin of 24 h grown mycelia on agar Dox medium. Over the incubation period (8 d), diameters of the colonies and of any clear zones of solubilization after peeling the membranes with the colonies were measured daily. Growth rates and rates of extension of solubilization zones were calculated by least squares regression. The pH values underneath the central part of the colonies were measured in duplicate using a combination pH electrode (Ingold ; Mettler-Toledo, Switzerland). Crystallography, SEM and X-ray microprobe analysis The solubilized zone was initially examined by light microscopy to detect the formation of crystals. These crystals were extracted from the agar by gently homogenizing the agar with 5 ml ddH O in a crystallizing dish. The crystals # were then allowed to settle, the aqueous phase was removed and the precipitated crystals were washed i3 with ddH O. # Samples of these crystals were mounted on double-sided carbon adhesive tape on aluminium stubs and these were dried overnight in a vacuum desiccator at room temperature. For SEM, the samples were sputter-coated for 5 min using a Polaron E5100 Series II cool sputter coated fitted with Au\Pd target before examination with a JEOL JSM-35 SEM. For energy dispersive X-ray microanalysis (EDXA), uncoated samples were used. Specimens were analysed for at least 100 s. Determination of oxalic, citric and gluconic acid 0n1 g of standard (BDH) calcium oxalate crystals, as well as newly-produced crystals after drying at 60m for 24 h were dissolved in 0n5 H SO in a boiling water bath for 15 min. # % After cooling and appropriate dilution, the samples were analysed by a HPLC system comprising a Waters (Watford, Herts, U.K.) 600E system controller, a Waters U6K pump and a Waters 717 plus autosampler controlled by Millipore (Waters) millennium chromatography software. The eluant used was 0n2 % orthophosphoric acidj1n0 % acetonitrile in MilliQ H O. The column used was a Spherisorb S5C8 # 25 cmi4n6 mm octyl bonded phase with a Techsphere 5C8 guard pre-column. The samples and eluant were pre-filtered through 0n45 µm membrane filters, and 20 µl duplicate samples were run at wavelength 200 nm for 15 min at room temperature. For estimation of the produced oxalic, citric and gluconic acids in liquid media, samples from the filtrate of a 10 d old culture were taken. For citric acid determination, the samples were diluted i10, filtered and analysed using HPLC as above. Oxalic acid was determined after extraction with tri n-butyl phosphate as described by Lapeyrie, Chilvers & Bhem (1987). A series of standard solutions of oxalic acid, citric acid and gluconic acid (sodium salts) (Sigma) (0–100 µ) was used. RESULTS Gypsum solubilization Gypsum solubilization by the fungi was monitored by the production of a clear and subsequently recrystallized zone
M. M. Gharieb and G. M. Gadd
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(a)
(b)
(c)
(d)
Fig. 1. Photographs of 9 cm diam. Petri dishes showing the solubilization of gypsum-amended nitrate-containing Czapek–Dox medium by (a) A. niger, (b) P. bilaii, (c) P. simplicissimum, and (d ) Pax. involutus. P. bilaii was grown for 10 d on 1n0 % (w\v) gypsum-containing medium whereas the other fungi were grown for 7 d on 0n5 % (w\v) gypsum-containing medium at 25m. All inoculations were on dialysis membrane overlaying the medium and the upper part of the membrane with the growing mycelium was cut away to show gypsum solubilization and the formation of calcium oxalate crystals.
under the growing colonies on solid medium. Fig. 1 shows the solubilization of 0n5 % (w\v) gypsum by A. niger, P. bilaii, Pax. involutus and P. simplicissimum. The first three species displayed the formation of new crystals inside a marginal clear zone, whereas P. simplicissimum showed a totally clear zone underneath the colony. A. niger formed concentric rings of new crystals that were more condensed than in Pax. involutus, but P. bilaii produced the smallest amount of crytstals. Despite there being no great differences in growth, gypsum dissolution was significantly affected by the form of the supplemented nitrogen source. While the ratio of gypsum solubilization to colony extension was low in ammonium-nitrogen medium, it was significantly higher in the presence of nitrate-nitrogen. On the other hand, Ph. chrysosporium and C. versicolor showed a low ability to solubilize gypsum in nitrate- or ammoniumcontaining medium (Table 1). In nitrate medium, the solubilization ratios (Rs\Rm) for A. niger, Pax. involutus, P. bilaii and P. simplicissimum were around unity, i.e. the rate of extension of the solubilized area approached the rate of fungal colony extension. This ratio was, however, very low when ammonium was used as a nitrogen source (Table 1). In ammonium–nitrogen medium, Pax. involutus and P. simpli-
cissimum displayed a solubilization ratio 20 % of that in nitrate–nitrogen, while A. niger and P. bilaii showed a solubilization ratio of 40 %. The presence of soluble sulphate affected the solubilization activity of only P. simplicissimum in ammonium–nitrogen medium with the solubilization ratio doubling despite a lack of effect on colony extension (Table 2). Crystal production The dissolved zones (inside the halo) were directly examined by light microscopy which confirmed the production of morphologically different crystals to the amorphous particles of gypsum. The relative abundance of these crystals was in the order A. niger Pax. involutus P. bilaii, whereas no crystals were detected in P. simplicissimum or the other two organisms. The prevalence of a certain shape of crystals was observed depending on the fungus and the age of the colony (Fig. 2). A. niger produced concentric rings that coincided with different morphological forms ; in the marginal area, where the most newly formed crystals were located, were tetragonal or prismatic ; in the middle, intermingled rod-like structures were dominant, whereas spherical structures were detected under
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Table 1. Ratio between colony growth rate on 0n5 % (w\v) gypsum (Rm) and the growth rate on gypsum-free (Rc) Czapek–Dox agar medium containing either nitrate or ammonium as a nitrogen source, in the absence (kS) or presence of sulphate (jS). The ratios between the solubilization rate on 0n5 % (w\v) gypsum (Rs) and colonial growth on the same concentration of gypsum (Rm) are also presented in the presence or absence of sulphur. Rates of growth and solubilization were calculated for three replicates over 8 d incubation at 25m. Ph. chrysosporium measurements were taken over only 4 d as a result of its fast growth rate. The pH values (means of two readings) at the centre of the plate underneath the growing colonies on gypsum-containing medium are also given Rm\Rc
Rs\Rm
pH
kS
jS
kS
jS
kS
jS
Nitrate A. niger C. versicolor Ph. chrysosporium P. bilaii P. simplicissimum Pax. involutus
1n07 0n91 1n10 1n07 1n03 0n98
1n09 0n78 1n10 0n99 0n79 0n98
0n86 0n0 0n0 1n34 1n02 0n95
0n85 0n00 0n00 1n32 1n02 0n99
2n7 4n1 3n8 4n8 7n9 5n4
2n7 4n0 3n8 4n6 8n0 6n8
Ammonium A. niger C. versicolor Ph. chrysosporium P. bilaii P. simplicissimum Pax. involutus
1n07 1n00 1n00 0n97 0n98 1n02
1n27 0n80 1n00 1n12 1n03 1n32
0n43 0n00 0n00 0n92 0n37 0n26
0n43 0n00 0n00 0n93 0n73 0n25
2n1 3n0 2n4 2n3 2n2 2n4
2n1 3n0 2n4 2n3 2n2 2n3
Table 2. Ratio between growth on ammonium-nitrogen (GAmm) and that on nitrate-nitrogen (GNit) Czapek–Dox agar medium in the absence (kS) or the presence of sulphate (jS) after incubation of the fungi for 8 d at 25m. The ratio between the solubilized zone on ammonium (SAmm) and solubilization on nitrate-containing (SNit) medium is also indicated. Five replicates were used for each measurement GAmm\GNit
A. niger C. versicolor Ph. chrysosporium P. bilaii P. simplicissimum Pax. involutus SXK2A
(a)
SAmm\SNit
kS
jS
kS
jS
0n87 0n36 1n0 0n56 0n56 0n87
0n87 0n31 1n0 0n62 0n56 0n75
0n43 0n0 0n0 0n39 0n20 0n24
0n44 0n0 0n0 0n44 0n40 0n19
the centre of the colony. Pax. involutus and P. bilaii showed an abundance of prismatic-like crystals over the whole of the recrystallized zone (Fig. 2). Crystals were extracted from the agar and examined by SEM (Fig. 3). This revealed that the prevalent crystals were tetragonal di-pyramids in Pax. involutus (Fig. 3 f ) and P. bilaii (Fig. 3 e), but in A. niger these crystals were also initially formed but then observed no change into plate-like structures resembling planar druses (Fig. 3 d ). X-ray microanalysis of individual gypsum particles showed peaks for calcium and sulphur. On the new crystals, the only peak present was calcium which indicated the release of sulphate (Fig. 4 a, b). After dissolution of dried samples of these crystals, HPLC analysis resulted in identical peaks occurring at 3n1 min, with such peaks matching those that result from both pure oxalic
(b)
(c)
Fig. 2. Photomicrographs of agar showing (a) gypsum particles before solubilization (b) the particles during solubilization and the early formation of new calcium oxalate crystals by P. bilaii, and (c) different shapes of calcium oxalate crystals in the marginal crystallization zone formed by A. niger. Bar l 200 µm.
acid and standard calcium oxalate crystals (Fig. 5), suggesting that the produced crystals are calcium oxalate. Oxalic, citric and gluconic acid analysis The organic acids, particularly oxalic, citric and gluconic,
M. M. Gharieb and G. M. Gadd
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(a)
(b)
(c)
(d )
(e)
(f )
Fig. 3. SEM of (a) gypsum particles and different forms of calcium oxalate crystals formed by (b–d ) A. niger (e) P. bilaii and ( f ) Pax. involutus. The crystals were extracted from the agar before preparation for SEM as described in Methods. Bars l 10 µm for (a–d ) ; 100 µm for (e, f ).
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(a) Ca
S
Ca
(b) Ca
Ca
Fig. 4. X-ray microanalysis of (a) gypsum (CaSO ;2H O) particles % # and (b) extracted crystals produced underneath growing colonies of P. bilaii after growth on nitrate-containing Czapek–Dox medium containing 0n5 % (w\v) gypsum for 8 d at 25m. Data shown are representative of at least five replicates. Identical peaks were also obtained for purified crystals from A. niger and Pax. involutus. (a)
(b)
(c)
2
4
6
8
Time (min)
Fig. 5. HPLC chromatograms showing (a) oxalic acid, (b) standard calcium oxalate crystals and (c) the new crystals formed by P. bilaii. A similar pattern of peaks was also observed for the crystals produced by A. niger and Pax. involutus. Five replicates for each treatment were carried out.
produced in liquid medium during growth of the experimental fungi with the different nitrogen sources, are shown in Table 3. It is clear that in nitrate-containing medium, oxalic acid was excreted by P. bilaii, A. niger, Pax. involutus and P. simplicissimum in high amounts which were 23n6p0n22, 15n4p0n8, 9n6p0n8 and 19n9p4n4 mmol l−" respectively. In the same medium, however, Ph. chrysosporium and C. versicolor produced much smaller amounts of oxalic acid : 1n9p0n1 and 2n7p0n3 mmol l−" respectively. In contrast, only small amounts ( µmol l−") of oxalic acid were produced in ammonium–nitrogen medium by all the fungi except A. niger, which produced 4n6 mmol l−". Citric acid was not detected in the medium filtrate of Ph. chrysosporium and C. versicolor, but small quantities were produced by the other fungi in both the media used. In Czapek–Dox agar, the initial pH value was 5n1. After fungal growth, the final pH values of the agar underneath the colonies were recorded (Table 1). There were no significant differences between the pH of the control (gypsum-free) and the 0n5 % gypsum-containing medium. The final pH values of ammonium–nitrogen medium were, however, significantly lower than the nitrate medium. The same difference was also observed in liquid medium (Table 3). DISCUSSION The results provide clear evidence for gypsum solubilization by the fungi examined with the dissolution process being favoured by the presence of nitrate as sole nitrogen source. In contrast, when ammonium was used as the nitrogen source, the fungi were unable to solubilize gypsum despite both nitrogen sources being used for fungal growth (Table 3). Utilization of both ammonium and nitrate by fungi is well known and when nitrogen is provided as ammonium nitrate (NH NO ) ammonium ions are utilized preferentially as a % $ result of repression of nitrate reductase by the ammonium ion (Griffin, 1994). In this study oxalic acid, rather than citric acid, was found to be excreted in large amounts in nitratecontaining medium. A stimulation of oxalic acid production in the presence of nitrate and inhibition by ammonium was previously reported for the plant pathogenic Sclerotium rolfsii (Kritzman, Chet & Henis, 1977) and certain mycorrhizal fungi (Lapeyrie et al., 1987). It was postulated that the ammonium ions inhibited glyoxalate dehydrogenase activity, which is involved in oxalate synthesis from glyoxalate. In certain lower plants, Meeuse & Campbell (1959) suggested an inhibition by nitrate of oxalic acid oxidase which breaks down oxalic acid to carbon dioxide and hydrogen peroxide. A greater decrease in pH was also detected after fungal growth in ammoniumcontaining medium, compared to that occurring in nitrate medium. A marked fall in pH generally results during growth on ammonium as sole N source (Carlile & Watkinson, 1994). Oxalate decarboxylase, which catalyses breakdown of oxalic acid to carbon dioxide and formate, has been isolated from a number of fungi. It was found that production of this enzyme by A. niger was non-inducible and pH dependent, only being synthesized if the medium was below pH 2n5 (Emiliani & Bekes, 1964). A stimulation of proton extrusion by ammonium ions has been documented in many instances (MacMillan, 1956 ; Holligan & Jennings, 1972 ; Smith & Raven, 1979 ; Roos
M. M. Gharieb and G. M. Gadd
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Table 3. pH values, .., and organic acid content of Czapek–Dox liquid medium containing either nitrate or ammonium as a nitrogen source. Three replicates of 50 ml liquid medium were inoculated by two discs (7 mm) from the margin of 7 d old colony and statically incubated for 10 d at 25m. The figures are meansp...
A. niger Nitrate Ammonium C. versicolor Nitrate Ammonium Ph. chrysosporium Nitrate Ammonium P. bilaii Nitrate Ammonium P. simplicissimum Nitrate Ammonium Pax. involutus Nitrate Ammonium
.. (mg ml−")
pH
Oxalic acid (mmol l−")
Citric acid ( µmol l−")
Gluconic acid ( µmol l−")
7n22p1n15 6n80p1n06
2n3 2n1
15n40p0n80 4n60p1n60
17n80p4n40 22n60p2n05
0n0 1n47p0n29
1n66p0n29 1n97p0n59
4n4 3n2
2n70p0n30 0n20p0n03
n.d.* n.d.*
0n0 1n55p0n16
3n40p0n10 2n10p0n55
4n5 3n3
1n90p0n10 0n67p0n02
n.d.* n.d.*
0n00 0n73p0n03
2n98p0n10 5n05p0n20
3n6 2n2
23n60p0n22 0n65p0n05
14n00p0n32 2n40p0n07
0n0 1n20p0n05
3n46p1n20 2n25p0n61
4n4 2n7
19n90p4n40 0n61p0n30
6n50p1n20 1n03p0n50
0n0 1n06p0n09
4n98p0n80 3n45p0n89
3n4 2n2
9n60p0n80 0n67p0n03
29n20p1n20 13n30p2n50
1n33p0n70 1n82p0n17
* Not detected.
& Lackner, 1984). The ability of some ectomycorrhizal fungi to solubilize certain mineral phosphates has been documented in the presence of ammonium. Moreover, a strain of Paxillus involutus displayed the ability to solubilize calcium phosphate using either ammonium or nitrate nitrogen (Lapeyrie et al., 1990). Other studies have stated that ammonium was necessary for increased inorganic phosphate (rock phosphate) solubilization by P. bilaii (Asea et al., 1988). These authors concluded that the solubilization process was directly related to the pH fall generated by P. bilaii and P. cf. fuscum. Illmer & Schinner (1995) also drew the same conclusion for the solubilization of hydroxyapatite and brushite (phosphate minerals) by Penicillium aurantiogriseum and Pseudomonas sp. Dixon-Hardy et al. (1998) found that the rate of solubilization of Zn (PO ) and Co (PO ) by A. niger decreased with $ %# $ %# decreasing nitrogen (as ammonium) concentration, and increased with decreasing nitrogen (as nitrate) concentration. Additionally, there is evidence for the involvement of organic acids, particularly, citric acid and oxalic acid, as well as the acidity resulting from ammonium incorporation in phosphate solubilization by P. bilaii (Cunningham & Kuiack, 1992). Parks et al. (1990) reported the involvement of oxalic acid and various isomers of itaconic acid in the solubilization of apatites by a Penicillium sp. Our study indicates that gypsum solubilization is accomplished by oxalic and citric acid rather than gluconic acid nor the acidity resulting from ammonium utilization. For the same fungus, and despite the significant difference in oxalic acid production, there was no great difference between the resulting acidity of nitrate- and ammonium-containing medium. Although the ability to degrade cellulosic materials by Ph. chrysosporium and C. versicolor and cause rotting, their inability to solubilize gypsum may be due to the restricted amount of oxalic acid produced. Previous studies have also reported a low production of oxalic acid by these rot-causing
fungi, and this has been attributed to the presence of an intracellular oxalate decarboxylase which decomposes oxalate to carbon dioxide and formate (Takao, 1965 ; Espejo & Agosin, 1991). Dutton et al. (1993) also demonstrated that there was little oxalate accumulation during active growth, whereas millimolar concentrations of oxalate were detected in culture medium during the stationary phase of Ph. chrysosporium and C. versicolor. It was recorded that these fungi produced oxalic acid under poor nutrient conditions particularly regarding carbon source (Dutton et al., 1993 ; Kuan & Tien, 1993). A synergistic interaction between oxalic acid and polygalacturonase was suggested and both degradation agents were reported to be induced by pectin (Green et al., 1995). Oxalic acid and citric acid are characterized by their acidity and complexing ability. Here, the process of gypsum solubilization by all the fungi tested except P. simplicissimum, was shown to proceed through two steps, firstly the formation of a clear zone, then the formation of new crystals. The abundance of crystals was related to the acidity of the medium as well as the ratio of oxalic acid to citric acid with the acidity of the medium determining whether the acid is in the protonated form or not for the chelation of calcium ions. In previous experiments, we found that the sodium salt of oxalic acid had no influence on gypsum solubilization and the formation of calcium oxalate, while oxalic acid exhibited a high ability to solubilize gypsum with the formation of calcium oxalate crystals (Gharieb et al., 1997). Additionally, the role of citric acid in gypsum solubilization should be considered since in a separate experiment we found that 50 µl of 0n5, 1, 5 and 10 m pure citric acid when added to 0n7 cm diam. wells in Czapek–Dox agar containing 0n5 % (w\v) natural gypsum produced 0n7p0n0, 0n8p0n0, 1n3p0n1 and 1n8p0n1 cm diam. clear zones respectively. Such an ability of 5 m gluconic acid for gypsum solubilization was not, however, detected. There was also a synergistic activity
Fungal solubilization of gypsum between citric and oxalic acid regarding gypsum solubilization. Using 50 µl of 5n0 m pure citric and oxalic acid added separately and in an equal mixture to 7 mm diam. wells, the diameters of solubilized areas were 1n4p0n1 and 1n5p0n1 cm for citric and oxalic acid, and 2n1p0n2 cm for the mixture. The production of a clear solubilized zone with P. simplicissimum could be attributable to either the dominance of citric acid for gypsum solubilization, since this fungus is known to excrete considerable amounts of citric acid ( 100 m) in the presence of insoluble metal oxides such as zinc oxide and industrial filter dust (Franz et al., 1991). These authors also reported that the pH range during the time of citric acid production by P. simplicissimum is neutral to slightly acidic. The formation of other soluble calcium compounds with other fungal metabolites and\or the accumulation of calcium ions inside the cells are also probable (Hodgkinson, 1977). Calcium oxalate crystals are known in two hydration states ; monohydrate (whewellite) and di- or polyhydrate (weddellite) with the availability of calcium affecting the morphology and hydration state. The crystals obtained here are morphologically identical to weddellite (Frey-Wyssling, 1981). This kind of calcium oxalate crystal occurs naturally in many fungi belonging to the Mucorales, Basidiomycetes and ectomycorrhizas (Arnott, 1995). In conclusion, our results confirm the mechanism of gypsum solubilization being primarily due to oxalic acid and\or citric acid production and further underline the potential role played by both free-living and symbiotic oxalic acid and citric acid-producing fungi in the biogeochemistry soil minerals. These findings may have further relevance for land reclamation and agricultural applications in providing soluble sulphate from gypsum for plant nutrition. In addition, the formation of insoluble calcium oxalate would immobilize potentially toxic calcium ions and protect both fungi and plants in the vicinity. MMG gratefully acknowledges financial support from the Royal Society (London) by the award of a Third World Research Fellowship. GMG also gratefully acknowledges financial support from the Biotechnology and Biological Sciences Research Council (SPC 02922 ; SPC 02812). Martin Kierans, of this Department, is thanked for assistance with the EM and X-ray microprobe analysis. REFERENCES Agnihotri, V. P. (1970). Solubilization of insoluble phosphates by some soil fungi isolated from nursery seedbeds. Canadian Journal of Microbiology 16, 877–880. Alva, A. K., Gasho, G. J. & Guang, Y. (1989). Gypsum material effects on peanut and soil calcium. Communications in Soil Science and Plant Analysis 20, 1727–1744. Arnott, H. J. (1995). Calcium oxalate in fungi. In Calcium Oxalate in Biological Systems (ed. S. R. Khan), pp. 73–111. CRC Press : Boca Raton. Asea, P. E. A., Kucey, R. M. N. & Stewart, J. W. B. (1988). Inorganic phosphate solubilization by two Penicillium species in solution culture and soil. Soil Biology and Biochemistry 20, 459–464. Buerkert, A. & Marschner, H. (1992). Calcium and temperature effects on seedling exudation and root-rot infection of common bean on an acid sandy soil. Plant and Soil 147, 293–303. Burgstaller, W. & Schinner, F. (1993). Leaching of metals with fungi. Journal of Biotechnology 27, 91–116. Cromack, K., Sollins, P., Graustein, W. C., Speidel, K., Todd, A. W., Psycher,
480 G., Li, C. Y. & Todd, R. L. (1979). Calcium oxalate accumulation and soil weathering in mats of the hypogeous fungus Hysterangium crassum. Soil Biology 11, 463–468. Cunningham, J. E. & Kuiack, C. (1992). Production of citric and oxalic acids and solubilization of calcium phosphates by Penicillium bilaii. Applied and Environmental Microbiology 58, 1451–1458. Dixon-Hardy, J. E., Karamushka, V. I., Gruzina, T. G., Nikovska, G. N., Sayer, J. A. & Gadd, G. M. (1998). Influence of the carbon, nitrogen and phosphorus source on the solubilization of insoluble metal compounds by Aspergillus niger. Mycological Research 102, 1050–1054. Douglas, J. & Singh, J. (1995). Investigating dry rot in buildings. Building Research and Information 23, 345–352. Dutton, M. V., Evans, C. S., Atkey, P. T. & Wood, D. A. (1993). Oxalate production by Basidiomycetes, including the white rot species Coriolus versicolor and Phanerochaete chrysosporium. Applied Microbiology and Biotechnology 39, 5–10. Ellington, A., Badawy, N. S. & Ganning, G. W. (1997). Testing gypsum requirements for dryland cropping on a red-brown earth. Australian Journal of Soil Research 35, 591–607. Emiliani, E. & Bekes, P. (1964). Enzymatic oxalate decarboxylation in Aspergillus niger. Archives of Biochemistry and Biophysics 105, 488–493. Espejo, E. & Agosin, E. (1991). Production and degradation of oxalic acid by brown rot fungi. Applied and Environmental Microbiology 57, 1980–1986. Franz, A., Burgstaller, W. & Schinner, F. (1991). Leaching with Penicillium simplicissimum : influence of metals and buffers on proton extrusion and citric acid production. Applied and Environmental Microbiology 57, 769–774. Frey-Wyssling, A. (1981). Crystallography of the two hydrates of crystalline calcium oxalate in plants. American Journal of Botany 68, 130–141. Gharieb, M. M., Sayer, J. A. & Gadd, G. M. (1997). Solubilization of natural gypsum (CaSO ;2H O) and the formation of calcium oxalate by Aspergillus % % niger and Serpula himantioides. Mycological Research 102, 825–830. Graustein, W. C., Cromack, K. & Sollins, P. (1977). Calcium oxalate : occurrence in soils and effect on nutrient and geochemical cycles. Science 198, 1252–1254. Green, F., Clausen, C. A., Kuster, T. A. & Highley, T. L. (1995). Induction of polygalacturonase and the formation of oxalic acid by pectin in brown-rot fungi. World Journal of Microbiology and Biotechnology 11, 519–524. Griffin, D. H. (1994). Fungal Physiology. Wiley-Liss : New York. Hodgkinson, A. (1977). Oxalic Acid in Biology and Medicine. Academic Press : New York. Holligan, P. M. & Jennings, D. H. (1972). The influence of different carbon and nitrogen sources on the accumulation of mannitol and arabitol. New Phytologist 71, 583–594. Hughes, M. N. & Poole, R. K. (1989). Metals and Micro-organisms. Chapman and Hall : London. Illmer, P. & Schinner, F. (1995). Solubilization of inorganic calcium phosphatessolubilization mechanisms. Soil Biology and Biochemistry 27, 257–263. Jones, D., Smith, B. F., Wilson, M. J. & Goodman, B. A. (1991). Phosphate solubilizing fungi in a Scottish upland soil. Mycological Research 95, 1090–1093. Jones, D., Wilson, M. J. & Tait, J. M. (1980). Weathering of basalt by Pertusaria corallina. Lichenologist 12, 277–289. Kant, S. & Kumar, R. (1992). Effect of gypsum, pyrite, pressmud and farmyard manure on soil properties and yield of rice (Oryza sativa). Indian Journal of Agricultural Sciences 62, 191–195. Kritzman, G., Chet, I. & Henis, Y. (1977). The role of oxalic acid in the pathogenic behaviour of Sclerotium rolfsii Sacc. Experimental Mycology 1, 280–285. Kuan, I. C. & Tien, M. (1993). Stimulation of Mn peroxidase activity : a possible role for oxalate in lignin biodegradation. Proceedings of the National Academy of Sciences of the U.S.A. 90, 1242–1246. Kucey, R. M. (1987). Increased phosphorus uptake by wheat and field beans inoculated with a phosphorus solubilizing Penicillum bilaii strain and with versicular-arbuscular mycorrhizal fungi. Applied and Environmental Microbiology 53, 2699–2703. Lapeyrie, F., Chilvers, G. A. & Bhem, C. A. (1987). Oxalic acid synthesis by the mycorrhizal fungus Paxillus involutus (Batsch ex Fr.)Fr. New Phytologist 106, 139–146. Lapeyrie, F., Ranger, J. & Vairelles, D. (1990). Phosphate solubilizing activity of ectomycorrhizal fungi in vitro. Canadian Journal of Botany 69, 342–346.
M. M. Gharieb and G. M. Gadd MacMillan, A. (1956). The entry of ammonia into fungal cells. Journal of Experimental Botany 7, 113–126. Malajczuk, N. & Cromack, K. Jr (1982). Accumulation of calcium oxalate in the mantle of ectomycorrhizal roots of Pinus radiata and Eucalyptus marginata. New Phytologist 92, 527–531. Meesue, B. J. & Campbell, J. M. (1959). An inhibitor of oxalic acid oxidase in beet extracts. Plant Physiology 34, 583–586. Morley, G. F., Sayer, J. A., Wilkinson, S. C., Gharieb, M. M. & Gadd, G. M. (1996). Fungal sequestration, mobilization and transformation of metals and metalloids. In Fungi and Environmental Change (ed. J. C. Frankland, N. Magan & G. M. Gadd), pp. 235–256. Cambridge University Press : Cambridge. Nelson, R. E. (1982). Carbonate and gypsum. In Methods of Soil Analysis. Part 2 – Chemical and Microbiological Properties (2nd edn), pp. 181–197. (ed. A. L. Page, R. H. & D. R. Kenney). American Society of Agronomy : Madison, Wisconsin, U.S.A. Parks, E. J., Olson, G. J., Brinckman, F. E. & Baldi, F. (1990). Characterization by high performance liquid chromatography (HPLC) of the solubilization of phosphorus in iron ore by a fungus. Journal of Industrial Microbiology 5, 183–190. Reading, C. L., Harrison, M. A. & Kvien, C. K. (1992). Aspergillus parasiticus growth and aflatoxin synthesis on florunner peanuts grown in gypsumsupplemented soil. Journal of Food Protection 56, 593–594. (Accepted 29 June 1998)
481 Roos, W. & Lackner, M. (1984). Relationship between proton extrusion and fluxes of ammonium ions and organic acids in Penicillium cyclopium. Journal of General Microbiology 130, 1007–1014. Sayer, J. A. & Gadd, G. M. (1997). Solubilization and transformation of insoluble metal compounds to insoluble metal oxalates by Aspergillus niger. Mycological Research 101, 653–661. Sayer, J. A., Raggett, S. L. & Gadd, G. M. (1995). Solubilization of insoluble metal compounds by soil fungi : development of a screening method for solubilizing ability and metal tolerance. Mycological Research 99, 987–993. Smith, F. A. & Raven, J. A. (1979). Intracellular pH and its regulation. Annual Review of Plant Physiology 30, 289–311. Takao, S. (1965). Organic acid production by Basidiomycetes. Applied Microbiology 13, 732–737. Wallace, A. (1994 a). Use of gypsum in soil where needed can make agriculture sustainable. Communications in Soil Science and Plant Analysis 25, 109–116. Wallace, A. (1994 b). Soil improvement to match 21st-century agriculture. Communications in Soil Science and Plant Analysis 25, 165–169. White, C., Sayer, J. A. & Gadd, G. M. (1997). Microbial solubilization and immobilization of toxic metals : key biogeochemical processes for treatment of contamination. FEMS Microbiology Reviews 20, 503–516.
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Influence of nitrogen source on the solubilization of natural gypsum (CaSO4 . 2H2O) and the formation of calcium oxalate by different oxalic and citric acid-producing fungi
M O H A M M E D M. G H A R I EB1 A N D G E O F F R E Y M. G A D D2* " Department of Botany, Faculty of Science, Menoufia University, Shebein El-Koom, Egypt # Department of Biological Sciences, University of Dundee, Dundee, DD1 4HN, Scotland, U.K.
The ability of six fungi to solubilize natural-occurring gypsum was tested in vitro. The solubilization process was monitored by the production of a clear zone (halo) around or underneath the growing colony on Czapek–Dox agar containing 0n5 % (w\v) gypsum (CaSO ;2H O). Aspergillus niger, Penicillium bilaii, P. simplicissimum and Paxillus involutus displayed differential solubilization activities % # depending on the supplemented nitrogen source. Colonies grown on nitrate-containing medium showed the ability to solubilize gypsum, but when ammonium (at equivalent nitrogen) was used there was a significant reduction in solubilization. It was found that liquid cultures of nitrate-grown fungi produced substantial amounts of oxalic acid, whereas in ammonium-containing medium oxalic acid was only detected in small amounts. The production of citric and gluconic acid under these experimental conditions was low in both media, although the involvement of citric acid in gypsum solubilization is possible. Coriolus versicolor and Phanaerochaete chrysosporium did not exhibit any solubilization activity in nitrate- or ammonium-containing medium. Additionally these two fungi excreted small quantities of oxalic acid in both media with no citric acid being produced in liquid medium. Concomitant with the solubilization process and inside the clear solubilized zone, A. niger, Pax. involutus and P. bilaii produced crystals of differing shapes and abundance depending on the fungal strain. No crystals were produced by P. simplicissimum. These crystals were identified as calcium oxalate based on HPLC analysis and energy-dispersive X-ray microanalysis. Abundance of these crystals was found to be correlated with both oxalic acid production and the acidity of the medium. It is concluded that gypsum solubilization was predominantly achieved by both oxalic acid, which was accompanied by formation of calcium oxalate crystals, and citric acid production rather than the acidity of the medium. The results are discussed in relation to the significance of such an activity in agricultural applications, e.g. land reclamation, as well as the possible roles played by these fungi in mineral cycling.
The process of mineral solubilization by heterotrophic microorganisms has been apparent for many years. Recently, this phenomenon has received attention regarding biotechnological applications for leaching and recovery of metals from low grade ores and biogeochemical cycling of nutrients (Franz, Burgstaller & Schinner, 1991 ; Burgstaller & Schinner, 1993 ; Morley et al., 1996 ; White, Sayer & Gadd, 1997). In agriculture, as a result of the essentiality of phosphorus for plant growth, attention has been paid to the solubilization of insoluble mineral phosphates by soil microorganisms and numerous studies have highlighted the significant role played by soil fungi (Kucey, 1987 ; Asea, Kucey & Stewart, 1988 ; Lapeyrie, Ranger & Vairelles, 1990 ; Jones et al., 1991). A commercial formulation of Penicillium bilaii spores has been registered in Canada to increase available phosphate to wheat (Cunningham & Kuiack, 1992). Different mechanisms of solubilization have been identified including proton excretion, and the production of organic acids, and other chelating metabolites (Agnihotri, 1970 ; Hughes & Poole, 1989 ; Lapeyrie et al., 1990 ; Sayer & Gadd, 1997). Cunningham & Kuiack
* Corresponding author.
(1992) demonstrated that oxalic and citric acid and acidity resulting from ammonium incorporation are significant components of laboratory culture phosphate solubilization by P. bilaii. Oxalic acid is a common metabolite produced by several free-living and symbiotic fungi, and is implicated in the weathering of soil minerals, wood decay, building destruction and solubilization of metal-containing insoluble compounds (Graustein, Cromack & Sollins, 1977 ; Cromack et al., 1979 ; Jones, Wilson & Tait, 1980 ; Malajczuk & Cromack, 1982 ; Jones et al., 1991 ; Douglas & Singh, 1995 ; Sayer & Gadd, 1997). The effectiveness of oxalic acid is attributed to its behaviour as a proton source and the chelating ability of the oxalate anion (Jones et al., 1980 ; Sayer & Gadd, 1997). The most widespread form of naturally-occurring insoluble sulphate is the dihydrated form of calcium sulphate, gypsum (CaSO ;2H O), which occurs in several soils particularly % # those in arid and semi-arid areas (Nelson, 1982). Gypsum is also a by-product of various manufacturing operations and is introduced in building construction. During the last few years the application of gypsum to soil has become attractive because of the resulting improvement in soil properties and the yield of certain crop plants (Alva, Gasho & Guang, 1989 ; Kant & Kumar, 1992 ; Burkert & Marschner, 1992 ; Reading,
Fungal solubilization of gypsum Harrison & Kvien, 1992 ; Ellington, Badawy & Ganning, 1997). Wallace (1994 a, b) reported that gypsum is a key ingredient of many agricultural soils because it can improve physical and chemical properties of soil thus making a better environment for plant growth. In a previous study, we have demonstrated the ability of Aspergillus niger and Serpula himantioides to solubilize gypsum and release sulphate (Gharieb, Sayer & Gadd, 1997). The aim of this study was to investigate the activity of a number of organic acid-producing fungi, particularly citric and oxalic acid, in gypsum solubilization, to characterize this phenomenon further, and to provide details about the probable mechanism(s) involved.
MATERIALS AND METHODS Organisms, media and culture conditions The fungi used were Aspergillus niger Tiegh. (ATCC 201373), Penicillium bilaii Chalab. (kindly obtained from Dr C. Kuiack, Philom Bios Inc., Saskatoon, Canada), P. simplicissimum (Oudem.) Thom (Dr D. Leake, Imperial College, London) Paxillus involutus (Batsch) Fr. (Dr J. Frankland, Institute of Terrestrial Ecology, Merelewood), Phanaerochaete chrysosporium Burds. (Dr N. White, University of Abertay, Dundee) and Coriolus versicolor (L.) Que! l. (Prof C. S. Evans, University of Westminster, London). All were maintained routinely on malt extract agar (MEA, Lab M) at 25 mC. For experiments Czapek–Dox liquid medium was used of the following composition (g l−" distilled water) : sucrose, 30 ; KH PO , 1 ; # % MgSO ;7H O, 0n5 ; KCl, 0n5 ; FeCl , 0n001. Two different % # $ nitrogen sources were used ; either NaNO or NH Cl at $ % concentrations of 2 and 1n27 g l−" respectively. Two 7 mm diam. discs from the margin of 7 d old colonies were used as an inoculum in triplicate cultures of 50 ml liquid medium. 15 g l−" agar (Lab M, No. 2) was used for preparation of solid Czapek–Dox medium. High purity natural gypsum (CaSO ;2H O) was obtained from R. G. Widdowson % # (Scarborough, Yorks, U.K.). The particles were powdered in a ball mill, and the composition and purity verified by powder X-ray diffraction using a Hilton–Brooks X-ray diffractometer fitted with a curved graphite monochromator, using Cu Kα primary radiation. The obtained powder was then passed through a 250 µm sieve. A stock suspension in sterile ddH O # was prepared after dry autoclaving (for 15 min at 121m) of a known weight. To obtain a constant thickness of agar, 10 ml aliquots of agar medium (in triplicate) were autoclaved, and after cooling to 55m and just before pouring, an aliquot volume from the stock gypsum suspension was added to obtain a final concentration of 0n5 % (w\v). Control (gypsum-free) as well as sulphur-free treatments (in which MgCl was used instead of # MgSO ;7H O) were also prepared. Before pouring into % # 90 mm diam. Petri dishes, the media were mixed well to ensure homogeneous distribution of gypsum particles. After setting of the agar and prior to inoculation, " 80 mm diam. discs of autoclaved sterile dialysis membrane (BDH, Poole, U.K.) were placed under aseptic conditions onto the surface of the agar in each Petri dish (Sayer, Raggett & Gadd, 1995). The membrane allowed passage of nutrients\metabolites between the fungus and the agar and provided a convenient means of
474 removing the mycelium. Agar medium was inoculated with 7 mm diam. discs of mycelium cut from the margin of 24 h grown mycelia on agar Dox medium. Over the incubation period (8 d), diameters of the colonies and of any clear zones of solubilization after peeling the membranes with the colonies were measured daily. Growth rates and rates of extension of solubilization zones were calculated by least squares regression. The pH values underneath the central part of the colonies were measured in duplicate using a combination pH electrode (Ingold ; Mettler-Toledo, Switzerland). Crystallography, SEM and X-ray microprobe analysis The solubilized zone was initially examined by light microscopy to detect the formation of crystals. These crystals were extracted from the agar by gently homogenizing the agar with 5 ml ddH O in a crystallizing dish. The crystals # were then allowed to settle, the aqueous phase was removed and the precipitated crystals were washed i3 with ddH O. # Samples of these crystals were mounted on double-sided carbon adhesive tape on aluminium stubs and these were dried overnight in a vacuum desiccator at room temperature. For SEM, the samples were sputter-coated for 5 min using a Polaron E5100 Series II cool sputter coated fitted with Au\Pd target before examination with a JEOL JSM-35 SEM. For energy dispersive X-ray microanalysis (EDXA), uncoated samples were used. Specimens were analysed for at least 100 s. Determination of oxalic, citric and gluconic acid 0n1 g of standard (BDH) calcium oxalate crystals, as well as newly-produced crystals after drying at 60m for 24 h were dissolved in 0n5 H SO in a boiling water bath for 15 min. # % After cooling and appropriate dilution, the samples were analysed by a HPLC system comprising a Waters (Watford, Herts, U.K.) 600E system controller, a Waters U6K pump and a Waters 717 plus autosampler controlled by Millipore (Waters) millennium chromatography software. The eluant used was 0n2 % orthophosphoric acidj1n0 % acetonitrile in MilliQ H O. The column used was a Spherisorb S5C8 # 25 cmi4n6 mm octyl bonded phase with a Techsphere 5C8 guard pre-column. The samples and eluant were pre-filtered through 0n45 µm membrane filters, and 20 µl duplicate samples were run at wavelength 200 nm for 15 min at room temperature. For estimation of the produced oxalic, citric and gluconic acids in liquid media, samples from the filtrate of a 10 d old culture were taken. For citric acid determination, the samples were diluted i10, filtered and analysed using HPLC as above. Oxalic acid was determined after extraction with tri n-butyl phosphate as described by Lapeyrie, Chilvers & Bhem (1987). A series of standard solutions of oxalic acid, citric acid and gluconic acid (sodium salts) (Sigma) (0–100 µ) was used. RESULTS Gypsum solubilization Gypsum solubilization by the fungi was monitored by the production of a clear and subsequently recrystallized zone
M. M. Gharieb and G. M. Gadd
475
(a)
(b)
(c)
(d)
Fig. 1. Photographs of 9 cm diam. Petri dishes showing the solubilization of gypsum-amended nitrate-containing Czapek–Dox medium by (a) A. niger, (b) P. bilaii, (c) P. simplicissimum, and (d ) Pax. involutus. P. bilaii was grown for 10 d on 1n0 % (w\v) gypsum-containing medium whereas the other fungi were grown for 7 d on 0n5 % (w\v) gypsum-containing medium at 25m. All inoculations were on dialysis membrane overlaying the medium and the upper part of the membrane with the growing mycelium was cut away to show gypsum solubilization and the formation of calcium oxalate crystals.
under the growing colonies on solid medium. Fig. 1 shows the solubilization of 0n5 % (w\v) gypsum by A. niger, P. bilaii, Pax. involutus and P. simplicissimum. The first three species displayed the formation of new crystals inside a marginal clear zone, whereas P. simplicissimum showed a totally clear zone underneath the colony. A. niger formed concentric rings of new crystals that were more condensed than in Pax. involutus, but P. bilaii produced the smallest amount of crytstals. Despite there being no great differences in growth, gypsum dissolution was significantly affected by the form of the supplemented nitrogen source. While the ratio of gypsum solubilization to colony extension was low in ammonium-nitrogen medium, it was significantly higher in the presence of nitrate-nitrogen. On the other hand, Ph. chrysosporium and C. versicolor showed a low ability to solubilize gypsum in nitrate- or ammoniumcontaining medium (Table 1). In nitrate medium, the solubilization ratios (Rs\Rm) for A. niger, Pax. involutus, P. bilaii and P. simplicissimum were around unity, i.e. the rate of extension of the solubilized area approached the rate of fungal colony extension. This ratio was, however, very low when ammonium was used as a nitrogen source (Table 1). In ammonium–nitrogen medium, Pax. involutus and P. simpli-
cissimum displayed a solubilization ratio 20 % of that in nitrate–nitrogen, while A. niger and P. bilaii showed a solubilization ratio of 40 %. The presence of soluble sulphate affected the solubilization activity of only P. simplicissimum in ammonium–nitrogen medium with the solubilization ratio doubling despite a lack of effect on colony extension (Table 2). Crystal production The dissolved zones (inside the halo) were directly examined by light microscopy which confirmed the production of morphologically different crystals to the amorphous particles of gypsum. The relative abundance of these crystals was in the order A. niger Pax. involutus P. bilaii, whereas no crystals were detected in P. simplicissimum or the other two organisms. The prevalence of a certain shape of crystals was observed depending on the fungus and the age of the colony (Fig. 2). A. niger produced concentric rings that coincided with different morphological forms ; in the marginal area, where the most newly formed crystals were located, were tetragonal or prismatic ; in the middle, intermingled rod-like structures were dominant, whereas spherical structures were detected under
Fungal solubilization of gypsum
476
Table 1. Ratio between colony growth rate on 0n5 % (w\v) gypsum (Rm) and the growth rate on gypsum-free (Rc) Czapek–Dox agar medium containing either nitrate or ammonium as a nitrogen source, in the absence (kS) or presence of sulphate (jS). The ratios between the solubilization rate on 0n5 % (w\v) gypsum (Rs) and colonial growth on the same concentration of gypsum (Rm) are also presented in the presence or absence of sulphur. Rates of growth and solubilization were calculated for three replicates over 8 d incubation at 25m. Ph. chrysosporium measurements were taken over only 4 d as a result of its fast growth rate. The pH values (means of two readings) at the centre of the plate underneath the growing colonies on gypsum-containing medium are also given Rm\Rc
Rs\Rm
pH
kS
jS
kS
jS
kS
jS
Nitrate A. niger C. versicolor Ph. chrysosporium P. bilaii P. simplicissimum Pax. involutus
1n07 0n91 1n10 1n07 1n03 0n98
1n09 0n78 1n10 0n99 0n79 0n98
0n86 0n0 0n0 1n34 1n02 0n95
0n85 0n00 0n00 1n32 1n02 0n99
2n7 4n1 3n8 4n8 7n9 5n4
2n7 4n0 3n8 4n6 8n0 6n8
Ammonium A. niger C. versicolor Ph. chrysosporium P. bilaii P. simplicissimum Pax. involutus
1n07 1n00 1n00 0n97 0n98 1n02
1n27 0n80 1n00 1n12 1n03 1n32
0n43 0n00 0n00 0n92 0n37 0n26
0n43 0n00 0n00 0n93 0n73 0n25
2n1 3n0 2n4 2n3 2n2 2n4
2n1 3n0 2n4 2n3 2n2 2n3
Table 2. Ratio between growth on ammonium-nitrogen (GAmm) and that on nitrate-nitrogen (GNit) Czapek–Dox agar medium in the absence (kS) or the presence of sulphate (jS) after incubation of the fungi for 8 d at 25m. The ratio between the solubilized zone on ammonium (SAmm) and solubilization on nitrate-containing (SNit) medium is also indicated. Five replicates were used for each measurement GAmm\GNit
A. niger C. versicolor Ph. chrysosporium P. bilaii P. simplicissimum Pax. involutus SXK2A
(a)
SAmm\SNit
kS
jS
kS
jS
0n87 0n36 1n0 0n56 0n56 0n87
0n87 0n31 1n0 0n62 0n56 0n75
0n43 0n0 0n0 0n39 0n20 0n24
0n44 0n0 0n0 0n44 0n40 0n19
the centre of the colony. Pax. involutus and P. bilaii showed an abundance of prismatic-like crystals over the whole of the recrystallized zone (Fig. 2). Crystals were extracted from the agar and examined by SEM (Fig. 3). This revealed that the prevalent crystals were tetragonal di-pyramids in Pax. involutus (Fig. 3 f ) and P. bilaii (Fig. 3 e), but in A. niger these crystals were also initially formed but then observed no change into plate-like structures resembling planar druses (Fig. 3 d ). X-ray microanalysis of individual gypsum particles showed peaks for calcium and sulphur. On the new crystals, the only peak present was calcium which indicated the release of sulphate (Fig. 4 a, b). After dissolution of dried samples of these crystals, HPLC analysis resulted in identical peaks occurring at 3n1 min, with such peaks matching those that result from both pure oxalic
(b)
(c)
Fig. 2. Photomicrographs of agar showing (a) gypsum particles before solubilization (b) the particles during solubilization and the early formation of new calcium oxalate crystals by P. bilaii, and (c) different shapes of calcium oxalate crystals in the marginal crystallization zone formed by A. niger. Bar l 200 µm.
acid and standard calcium oxalate crystals (Fig. 5), suggesting that the produced crystals are calcium oxalate. Oxalic, citric and gluconic acid analysis The organic acids, particularly oxalic, citric and gluconic,
M. M. Gharieb and G. M. Gadd
477
(a)
(b)
(c)
(d )
(e)
(f )
Fig. 3. SEM of (a) gypsum particles and different forms of calcium oxalate crystals formed by (b–d ) A. niger (e) P. bilaii and ( f ) Pax. involutus. The crystals were extracted from the agar before preparation for SEM as described in Methods. Bars l 10 µm for (a–d ) ; 100 µm for (e, f ).
Fungal solubilization of gypsum
478
(a) Ca
S
Ca
(b) Ca
Ca
Fig. 4. X-ray microanalysis of (a) gypsum (CaSO ;2H O) particles % # and (b) extracted crystals produced underneath growing colonies of P. bilaii after growth on nitrate-containing Czapek–Dox medium containing 0n5 % (w\v) gypsum for 8 d at 25m. Data shown are representative of at least five replicates. Identical peaks were also obtained for purified crystals from A. niger and Pax. involutus. (a)
(b)
(c)
2
4
6
8
Time (min)
Fig. 5. HPLC chromatograms showing (a) oxalic acid, (b) standard calcium oxalate crystals and (c) the new crystals formed by P. bilaii. A similar pattern of peaks was also observed for the crystals produced by A. niger and Pax. involutus. Five replicates for each treatment were carried out.
produced in liquid medium during growth of the experimental fungi with the different nitrogen sources, are shown in Table 3. It is clear that in nitrate-containing medium, oxalic acid was excreted by P. bilaii, A. niger, Pax. involutus and P. simplicissimum in high amounts which were 23n6p0n22, 15n4p0n8, 9n6p0n8 and 19n9p4n4 mmol l−" respectively. In the same medium, however, Ph. chrysosporium and C. versicolor produced much smaller amounts of oxalic acid : 1n9p0n1 and 2n7p0n3 mmol l−" respectively. In contrast, only small amounts ( µmol l−") of oxalic acid were produced in ammonium–nitrogen medium by all the fungi except A. niger, which produced 4n6 mmol l−". Citric acid was not detected in the medium filtrate of Ph. chrysosporium and C. versicolor, but small quantities were produced by the other fungi in both the media used. In Czapek–Dox agar, the initial pH value was 5n1. After fungal growth, the final pH values of the agar underneath the colonies were recorded (Table 1). There were no significant differences between the pH of the control (gypsum-free) and the 0n5 % gypsum-containing medium. The final pH values of ammonium–nitrogen medium were, however, significantly lower than the nitrate medium. The same difference was also observed in liquid medium (Table 3). DISCUSSION The results provide clear evidence for gypsum solubilization by the fungi examined with the dissolution process being favoured by the presence of nitrate as sole nitrogen source. In contrast, when ammonium was used as the nitrogen source, the fungi were unable to solubilize gypsum despite both nitrogen sources being used for fungal growth (Table 3). Utilization of both ammonium and nitrate by fungi is well known and when nitrogen is provided as ammonium nitrate (NH NO ) ammonium ions are utilized preferentially as a % $ result of repression of nitrate reductase by the ammonium ion (Griffin, 1994). In this study oxalic acid, rather than citric acid, was found to be excreted in large amounts in nitratecontaining medium. A stimulation of oxalic acid production in the presence of nitrate and inhibition by ammonium was previously reported for the plant pathogenic Sclerotium rolfsii (Kritzman, Chet & Henis, 1977) and certain mycorrhizal fungi (Lapeyrie et al., 1987). It was postulated that the ammonium ions inhibited glyoxalate dehydrogenase activity, which is involved in oxalate synthesis from glyoxalate. In certain lower plants, Meeuse & Campbell (1959) suggested an inhibition by nitrate of oxalic acid oxidase which breaks down oxalic acid to carbon dioxide and hydrogen peroxide. A greater decrease in pH was also detected after fungal growth in ammoniumcontaining medium, compared to that occurring in nitrate medium. A marked fall in pH generally results during growth on ammonium as sole N source (Carlile & Watkinson, 1994). Oxalate decarboxylase, which catalyses breakdown of oxalic acid to carbon dioxide and formate, has been isolated from a number of fungi. It was found that production of this enzyme by A. niger was non-inducible and pH dependent, only being synthesized if the medium was below pH 2n5 (Emiliani & Bekes, 1964). A stimulation of proton extrusion by ammonium ions has been documented in many instances (MacMillan, 1956 ; Holligan & Jennings, 1972 ; Smith & Raven, 1979 ; Roos
M. M. Gharieb and G. M. Gadd
479
Table 3. pH values, .., and organic acid content of Czapek–Dox liquid medium containing either nitrate or ammonium as a nitrogen source. Three replicates of 50 ml liquid medium were inoculated by two discs (7 mm) from the margin of 7 d old colony and statically incubated for 10 d at 25m. The figures are meansp...
A. niger Nitrate Ammonium C. versicolor Nitrate Ammonium Ph. chrysosporium Nitrate Ammonium P. bilaii Nitrate Ammonium P. simplicissimum Nitrate Ammonium Pax. involutus Nitrate Ammonium
.. (mg ml−")
pH
Oxalic acid (mmol l−")
Citric acid ( µmol l−")
Gluconic acid ( µmol l−")
7n22p1n15 6n80p1n06
2n3 2n1
15n40p0n80 4n60p1n60
17n80p4n40 22n60p2n05
0n0 1n47p0n29
1n66p0n29 1n97p0n59
4n4 3n2
2n70p0n30 0n20p0n03
n.d.* n.d.*
0n0 1n55p0n16
3n40p0n10 2n10p0n55
4n5 3n3
1n90p0n10 0n67p0n02
n.d.* n.d.*
0n00 0n73p0n03
2n98p0n10 5n05p0n20
3n6 2n2
23n60p0n22 0n65p0n05
14n00p0n32 2n40p0n07
0n0 1n20p0n05
3n46p1n20 2n25p0n61
4n4 2n7
19n90p4n40 0n61p0n30
6n50p1n20 1n03p0n50
0n0 1n06p0n09
4n98p0n80 3n45p0n89
3n4 2n2
9n60p0n80 0n67p0n03
29n20p1n20 13n30p2n50
1n33p0n70 1n82p0n17
* Not detected.
& Lackner, 1984). The ability of some ectomycorrhizal fungi to solubilize certain mineral phosphates has been documented in the presence of ammonium. Moreover, a strain of Paxillus involutus displayed the ability to solubilize calcium phosphate using either ammonium or nitrate nitrogen (Lapeyrie et al., 1990). Other studies have stated that ammonium was necessary for increased inorganic phosphate (rock phosphate) solubilization by P. bilaii (Asea et al., 1988). These authors concluded that the solubilization process was directly related to the pH fall generated by P. bilaii and P. cf. fuscum. Illmer & Schinner (1995) also drew the same conclusion for the solubilization of hydroxyapatite and brushite (phosphate minerals) by Penicillium aurantiogriseum and Pseudomonas sp. Dixon-Hardy et al. (1998) found that the rate of solubilization of Zn (PO ) and Co (PO ) by A. niger decreased with $ %# $ %# decreasing nitrogen (as ammonium) concentration, and increased with decreasing nitrogen (as nitrate) concentration. Additionally, there is evidence for the involvement of organic acids, particularly, citric acid and oxalic acid, as well as the acidity resulting from ammonium incorporation in phosphate solubilization by P. bilaii (Cunningham & Kuiack, 1992). Parks et al. (1990) reported the involvement of oxalic acid and various isomers of itaconic acid in the solubilization of apatites by a Penicillium sp. Our study indicates that gypsum solubilization is accomplished by oxalic and citric acid rather than gluconic acid nor the acidity resulting from ammonium utilization. For the same fungus, and despite the significant difference in oxalic acid production, there was no great difference between the resulting acidity of nitrate- and ammonium-containing medium. Although the ability to degrade cellulosic materials by Ph. chrysosporium and C. versicolor and cause rotting, their inability to solubilize gypsum may be due to the restricted amount of oxalic acid produced. Previous studies have also reported a low production of oxalic acid by these rot-causing
fungi, and this has been attributed to the presence of an intracellular oxalate decarboxylase which decomposes oxalate to carbon dioxide and formate (Takao, 1965 ; Espejo & Agosin, 1991). Dutton et al. (1993) also demonstrated that there was little oxalate accumulation during active growth, whereas millimolar concentrations of oxalate were detected in culture medium during the stationary phase of Ph. chrysosporium and C. versicolor. It was recorded that these fungi produced oxalic acid under poor nutrient conditions particularly regarding carbon source (Dutton et al., 1993 ; Kuan & Tien, 1993). A synergistic interaction between oxalic acid and polygalacturonase was suggested and both degradation agents were reported to be induced by pectin (Green et al., 1995). Oxalic acid and citric acid are characterized by their acidity and complexing ability. Here, the process of gypsum solubilization by all the fungi tested except P. simplicissimum, was shown to proceed through two steps, firstly the formation of a clear zone, then the formation of new crystals. The abundance of crystals was related to the acidity of the medium as well as the ratio of oxalic acid to citric acid with the acidity of the medium determining whether the acid is in the protonated form or not for the chelation of calcium ions. In previous experiments, we found that the sodium salt of oxalic acid had no influence on gypsum solubilization and the formation of calcium oxalate, while oxalic acid exhibited a high ability to solubilize gypsum with the formation of calcium oxalate crystals (Gharieb et al., 1997). Additionally, the role of citric acid in gypsum solubilization should be considered since in a separate experiment we found that 50 µl of 0n5, 1, 5 and 10 m pure citric acid when added to 0n7 cm diam. wells in Czapek–Dox agar containing 0n5 % (w\v) natural gypsum produced 0n7p0n0, 0n8p0n0, 1n3p0n1 and 1n8p0n1 cm diam. clear zones respectively. Such an ability of 5 m gluconic acid for gypsum solubilization was not, however, detected. There was also a synergistic activity
Fungal solubilization of gypsum between citric and oxalic acid regarding gypsum solubilization. Using 50 µl of 5n0 m pure citric and oxalic acid added separately and in an equal mixture to 7 mm diam. wells, the diameters of solubilized areas were 1n4p0n1 and 1n5p0n1 cm for citric and oxalic acid, and 2n1p0n2 cm for the mixture. The production of a clear solubilized zone with P. simplicissimum could be attributable to either the dominance of citric acid for gypsum solubilization, since this fungus is known to excrete considerable amounts of citric acid ( 100 m) in the presence of insoluble metal oxides such as zinc oxide and industrial filter dust (Franz et al., 1991). These authors also reported that the pH range during the time of citric acid production by P. simplicissimum is neutral to slightly acidic. The formation of other soluble calcium compounds with other fungal metabolites and\or the accumulation of calcium ions inside the cells are also probable (Hodgkinson, 1977). Calcium oxalate crystals are known in two hydration states ; monohydrate (whewellite) and di- or polyhydrate (weddellite) with the availability of calcium affecting the morphology and hydration state. The crystals obtained here are morphologically identical to weddellite (Frey-Wyssling, 1981). This kind of calcium oxalate crystal occurs naturally in many fungi belonging to the Mucorales, Basidiomycetes and ectomycorrhizas (Arnott, 1995). In conclusion, our results confirm the mechanism of gypsum solubilization being primarily due to oxalic acid and\or citric acid production and further underline the potential role played by both free-living and symbiotic oxalic acid and citric acid-producing fungi in the biogeochemistry soil minerals. These findings may have further relevance for land reclamation and agricultural applications in providing soluble sulphate from gypsum for plant nutrition. In addition, the formation of insoluble calcium oxalate would immobilize potentially toxic calcium ions and protect both fungi and plants in the vicinity. MMG gratefully acknowledges financial support from the Royal Society (London) by the award of a Third World Research Fellowship. GMG also gratefully acknowledges financial support from the Biotechnology and Biological Sciences Research Council (SPC 02922 ; SPC 02812). Martin Kierans, of this Department, is thanked for assistance with the EM and X-ray microprobe analysis. REFERENCES Agnihotri, V. P. (1970). Solubilization of insoluble phosphates by some soil fungi isolated from nursery seedbeds. Canadian Journal of Microbiology 16, 877–880. Alva, A. K., Gasho, G. J. & Guang, Y. (1989). Gypsum material effects on peanut and soil calcium. Communications in Soil Science and Plant Analysis 20, 1727–1744. Arnott, H. J. (1995). Calcium oxalate in fungi. In Calcium Oxalate in Biological Systems (ed. S. R. Khan), pp. 73–111. CRC Press : Boca Raton. Asea, P. E. A., Kucey, R. M. N. & Stewart, J. W. B. (1988). Inorganic phosphate solubilization by two Penicillium species in solution culture and soil. Soil Biology and Biochemistry 20, 459–464. Buerkert, A. & Marschner, H. (1992). Calcium and temperature effects on seedling exudation and root-rot infection of common bean on an acid sandy soil. Plant and Soil 147, 293–303. Burgstaller, W. & Schinner, F. (1993). Leaching of metals with fungi. Journal of Biotechnology 27, 91–116. Cromack, K., Sollins, P., Graustein, W. C., Speidel, K., Todd, A. W., Psycher,
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481 Roos, W. & Lackner, M. (1984). Relationship between proton extrusion and fluxes of ammonium ions and organic acids in Penicillium cyclopium. Journal of General Microbiology 130, 1007–1014. Sayer, J. A. & Gadd, G. M. (1997). Solubilization and transformation of insoluble metal compounds to insoluble metal oxalates by Aspergillus niger. Mycological Research 101, 653–661. Sayer, J. A., Raggett, S. L. & Gadd, G. M. (1995). Solubilization of insoluble metal compounds by soil fungi : development of a screening method for solubilizing ability and metal tolerance. Mycological Research 99, 987–993. Smith, F. A. & Raven, J. A. (1979). Intracellular pH and its regulation. Annual Review of Plant Physiology 30, 289–311. Takao, S. (1965). Organic acid production by Basidiomycetes. Applied Microbiology 13, 732–737. Wallace, A. (1994 a). Use of gypsum in soil where needed can make agriculture sustainable. Communications in Soil Science and Plant Analysis 25, 109–116. Wallace, A. (1994 b). Soil improvement to match 21st-century agriculture. Communications in Soil Science and Plant Analysis 25, 165–169. White, C., Sayer, J. A. & Gadd, G. M. (1997). Microbial solubilization and immobilization of toxic metals : key biogeochemical processes for treatment of contamination. FEMS Microbiology Reviews 20, 503–516.