Jacqueline A. Sayer

Solubilization and transformation of insoluble inorganic metal compounds to insoluble metal oxalates by Aspergillus niger

Aspergillus niger, a fungus capable of citric and oxalic acid production, was grown on solid medium amended with selected insoluble metal compounds (ZnO, Zn3(PO4)2 and Co3(PO4)2). While A. niger was able to grow on and solubilize all three metal compounds, a crystalline precipitate was observed to form eventually in the clear zone of solubilization under and around the colony. These crystals were extracted from the agar, examined by SEM and X-ray micro-analysis and identified as metal oxalates by performance hplc and gas chromatography–mass spectrometry. The ability of A. niger to precipitate other metal oxalates was investigated with cadmium, copper and manganese phosphates and strontium nitrate. Metal oxalates were again produced and it was found that the different metals resulted in different and characteristic crystal morphologies. A. niger is therefore capable of transforming inorganic insoluble metal compounds into organic insoluble metal compounds. The significance of this process as a means of toxic metal immobilization is discussed.

LEAD MINERAL TRANSFORMATION BY FUNGI

Pyromorphite (Pb5(PO4)3Cl), the most stable lead mineral under a wide range of geochemical conditions [1], can form in urban and industrially contaminated soils [2] [3] [4] [5]. It has been suggested that the low solubility of this mineral could reduce the bioavailability of lead, and several studies have advocated pyromorphite formation as a remediation technique for lead-contaminated land [3] [5] [6], if necessary using addition of phosphate [6]. Many microorganisms can, however, make insoluble soil phosphate bioavailable [7] [8] [9] [10], and the solubilisation of insoluble metal phosphates by free-living and symbiotic fungi has been reported [11] [12] [13] [14] [15]. If pyromorphite can be solubilised by microbial phosphate-solubilising mechanisms, the question arises of what would happen to the released lead. We have now clearly demonstrated that pyromorphite can be solubilised by organic-acid-producing fungi, for example Aspergillus niger, and that plants grown with pyromorphite as sole phosphorus source take up both phosphorus and lead. We have also discovered the production of lead oxalate dihydrate by A. niger during pyromorphite transformation, which is the first recorded biogenic formation of this mineral. These mechanisms of lead solubilisation, or its immobilisation as a novel lead oxalate, have significant implications for metal mobility and transfer to other environmental compartments and organisms. The importance of considering microbial processes when developing remediation techniques for toxic metals in soils is therefore emphasised.

Solubilization of insoluble metal compounds by soil fungi: development of a screening method for solubilizing ability and metal tolerance

Two ubiquitous organic acid-producing soil fungi, Aspergillus niger and Penicillium simplicissimum , were screened for the ability to solubilize eight insoluble metal compounds: Al 2 O 3 , Al x (PO 4 ) x , CaCO 3 , Ca 3 (PO 4 ) 2 , Co 3 (PO 4 ) 2 , Mn x (PO 4 ) x , ZnO and Zn 3 (PO 4 ) 2 . From these, three were selected as test metal compounds for the screening of species, strains and isolates for solubilization properties: ZnO, Zn 3 (PO 4 ) 2 and Co 3 (PO 4 ) 2 . These compounds were used effectively to screen a further 56 strains of fungi, including soil isolates, for solubilizing activity (as revealed by the appearance of clear zones of solubilization around colonies) and metal tolerance (by comparison of colony growth rates). Ratios of the colony growth rate in the presence of a given metal compound ( R m ) to the control colony growth rate ( R c ), and the rate of extension of the clear zone of solubilization ( R s ) in relation to the growth rate of that colony ( R m ), were useful in characterizing these properties. Almost one-third of the natural isolates screened were able to solubilize at least one of ZnO, Zn 3 (PO 4 ) 2 and Co 3 (PO 4 ) 2 , with five strains (out of 56) being capable of solubilizing all three. Cobalt phosphate was the most toxic of the three compounds; zinc phosphate was the least toxic, and was most resistant to solubilization. Zinc oxide was the most readily solubilized compound. In addition to screening for solubilizing abilities and metal tolerance, the method described can also be used for comparison of species and strains to obtain isolates with specific properties for possible biotechnological use and also to examine natural fungal populations. The incorporation of selected metal compounds in the test medium may also be used to assess responses to specific metals and their compounds as a preclude to physiological studies.

Solubilization of natural gypsum (CaSO4.2H2O) and the formation of calcium oxalate by Aspergillus niger and Serpula himantioides

The natural dihydrate form of calcium sulphate, CaSO 4 .2H 2 O (gypsum), particularly found in gypsiferous soils and in certain building constructions, was found to be effectively solubilized by both Aspergillus niger and Serpula himantioides . When the fungi were grown on malt extract agar (MEA) medium amended with up to 1% (w/v) of gypsum, solubilization activity was evident by the appearance of a clear zone (haol) around and/or under the growing colonies. The haloes were found subsequently to enclose concentric rings of newly formed crystalline precipitate. The pH profile of the medium underneath the growing mycelium of A. niger was not affected by the presence of gypsum, while the S. himantioides pH profile displayed a slight reduction in pH values when grown on gypsum-containing media compared with the control. Electron microscopy and X-ray microanalysis of the crystals revealed the formation of calcium-containing crystals with the characteristic morphology typical of different forms of calcium oxalate. The complexing activity of fungal-produced organic acids with calcium was suggested to be the prevalent mechanism of solubilization with the production of oxalic acid resulting in precipitation of oxalate. Such activity resulted in the release of available sulphate which increased over the incubation period. The concentrations of sulphate released from 0.5% (w/v) gypsum after growth of A. niger and S. himantioides for 8 d were 14·7±0·7 and 7·4±0·9 μmol cm -3 respectively. These results are discussed in the light of their relevance to land reclamation and plant nutrition, as well as the weathering of building materials containing gypsum.

Solubilisation of some naturally occurring metal-bearing minerals, limescale and lead phosphate by Aspergillus niger

The ability of the soil fungus Aspergillus niger to tolerate and solubilise seven naturally occurring metal-bearing minerals, limescale and lead phosphate was investigated. A. niger was able to solubilise four of the test insoluble compounds when incorporated into solid medium: cuprite (CuO2), galena (PbS), rhodochrosite (Mn(CO3)x) and limescale (CaCO3). A. niger was able to grow on all concentrations of all the test compounds, whether solubilisation occurred or not, with no reduction in growth rate from the control. In some cases, stimulation of growth occurred, most marked with the phosphate-containing mineral, apatite. Precipitation of insoluble copper and manganese oxalate crystals under colonies growing on agar amended with cuprite and rhodochrosite was observed after 1-2 days growth at 25 degrees C. This process of oxalate formation represents a reduction in bioavailability of toxic cations, and could represent an important means of toxic metal immobilisation of physiological and environmental significance.

Binding of cobalt and zinc by organic acids and culture filtrates of Aspergillus niger grown in the absence or presence of insoluble cobalt or zinc phosphate

The ability of commercially-available citric, oxalic and gluconic acids to bind Co 2+ and Zn 2+ was investigated and compared with culture filtrates from Aspergillus niger , a fungus capable of citric, gluconic and oxalic acid production, grown in the presence and absence of cobalt or zinc phosphate. This work demonstrated that citric and oxalic acid and the A. niger culture filtrates can bind Co 2+ and Zn 2+ and in some cases, the culture filtrates were more efficient than commercial organic acids. Gluconic acid did not bind Co 2+ or Zn 2+ under the conditions used in this study. The presence of insoluble metal phosphates in the growth medium was found to markedly influence the production of organic acids and, while large concentrations of gluconic acid were produced in the presence of Co 3 (PO 4)2 , the culture filtrate was unable to bind Zn 2+ . The production of oxalic acid by A. niger when grown in the presence of Zn 3 (PO 4 ) 2 led to the precipitation of insoluble zinc oxalate, a phenomenon with implications for metal tolerance and toxicity. The significance of these findings in relation to the environmental mobility of metals and phosphate, and the role of fungi in such transformations are discussed.

Inhibition of H+ efflux from Saccharomyces cerevisiae by insoluble metal phosphates and protection by calcium and magnesium: inhibitory effects a result of soluble metal cations?

Insoluble metal phosphates were capable of inhibitory effects on plasma membrane H + -ATPase-mediated glucose-dependent H + efflux from Saccharomyces cerevisiae . The relative toxicities of the different compounds used was Zn > Co which was the same order as that obtained for soluble chlorides of these metals, although the toxicity of the latter was manifest at lower concentrations. Inhibitory effects of the metal phosphates were dependent on the amount in suspension and increased with increasing preincubation time. The inhibitory effect of toxic metal compounds on glucose-dependent H + efflux could be reduced or prevented by the addition of chlorides of calcium and magnesium, calcium being more effective than magnesium. The relative protective effects of Ca and Mg were similar for both soluble and insoluble compounds, and Ca could neutralize the influence of both cobalt and zinc phosphates. Although toxic effects could result from the solubilization of the insoluble metal phosphate compounds as a result of H + efflux releasing potentially toxic metal cations, there was no difference in the amount of metal cations released whether glucose was present or absent from the treatments. In addition, it was found that significant concentrations of soluble Co 2+ and Zn 2+ were released into metal phosphate suspensions in the absence or presence of cells with equilibrium concentrations in cell free suspensions (attained after 10–20 min) being about 60 m for Zn 3 (PO 4 ) 2 and about 65 m for Co 3 (PO 4 ) 2 . In the presence of cells, a lo equilibrium concentration was attained in the presence of Zn, probably due to uptake by glucose-dependent and -independent processes. Such uptake was not observed for released Co 2+ and could account for the higher toxicity of zinc phosphate compared to cobalt phosphate. Although some direct interactions with the insoluble phosphates must have been involved in overall inhibitory effects, it is concluded that a significant proportion of toxic effects resulted from free metal cations in solution, with protective effects of Ca and Mg resulting from competitive and stabilizing interactions at the cell surface.

Influence of the carbon, nitrogen and phosphorus source on the solubilization of insoluble metal compounds by Aspergillus niger

The effects of varying carbon (glucose), nitrogen ((NH 4 ) 2 SO 4 , KNO 3 ) and phosphate (KH 2 PO 4 ) source on solubilization of insoluble Co 3 (PO 4 ) 2 . 8H 2 O, Zn 3 (PO 4 ) 2 . 2H 2 O and ZnO by the soil fungus Aspergillus niger were assessed. Solubilization activity was quantified by measuring the clear zones produced around colonies of A. niger growing on solidified mineral salts medium amended with the insoluble metal compounds. Effects of nutrient variation on solubilizing properties were compared using ratios of colony growth rate on the metal compounds ( R m ) to control growth rate ( R c ) and the rate of extension of the zone of solubilization ( R s ) compared to the colony growth rate on the metal compound ( R m ), i.e. R m : R c and R s : R m . Ratios of solubilization rate to growth rate ( R s : R m ) on all the compounds decreased with decreasing glucose concentration; there was no solubilization of ZnO below 60 m m glucose and no solubilization of the metal phosphates below 6 m m glucose. Reducing the concentration of ammonium sulphate in the growth medium decreased R s : R m but these values were increased when the nitrogen source was nitrate. Reducing the phosphate concentration increased solubilization of Co 3 (PO 4 ) 2 but reduced solubilization of Zn 3 (PO 4 ) 2 . These findings demonstrate that manipulation of carbon, nitrogen and phosphate sources in the growth medium, and variation of the form of the nutrient source, can be used to alter the solubilizing ability of A. niger . Whilst, in the natural environment, this response to different nutrient sources allows optimal exploitation of resources, the potential to manipulate nutrients for maximum solubilizing ability may prove beneficial for the optimization of the solubilization of metal compounds with respect to the bioremediation of metal-contaminated wastes and polluted ecosystems. It could also prove useful in other biotechnological applications such as metal recycling and extraction of metals from low-grade ores.

Abstracts Of Papers Presented at A joint UK — Hebrew University Of Jerusalem (HUJ) Seminar On

S OF PAPERS PRESENTED AT A JOINT UK HEBREW UNIVERSITY OF JERUSALEM (HUJ) SEMINAR ON AGRICULTURE AND THE CLEAN E N V I R O N M E N T