Studies on mineral phosphate solubilization by cyanobacteria <Emphasis Type="Italic">Westiellopsis</Emphasis> and <Emphasis Type="Italic">Anabaena</Emphasis>

Two diazotrophic cyanobacteria, Westiellopsis prolifica and Anabaena variabilis were evaluated for elucidating the possible mechanism of mineral phosphate solubilization. Phosphate starved cyanobacteria evaluated for the presence of organic acids, extracellular compounds or enzymes that might have been produced and promoted the mineral phosphate solubilization with Mussorie Rock Phosphate and Tricalcium Phosphate as substrates. Both the cultures did not reveal production of organic acids throughout the incubation period when checked for decrease in media pH of the media and thin layer chromatography. Thin layer chromatography of culture filtrates showed the presence of hydrocarbon like compound. Further analysis of the culture filtrates with gas liquid chromatography, a single peak near to the retention time of 7.6 was observed in all extracts of culture filtrates irrespective of phosphate source. UV-visible spectra of culture filtrates revealed the absorption maxima of 276 nm. Gas chromatographic-mass spectrometric analysis of culture filtrates showed most intense peak in the electron impact (EI) ionization was at m/z 149 and molecular ion peaks at m/z 207 and 167, inferring the presence of phthalic acid. Among the mechanisms in mineral phosphate solubilization, it was evident that these cyanobacteria used phthalic acid as possible mode of P solubilization.     

Bioleaching of Uranium From Egyptian Rocks Using Native Actinomycete Strains

Bioleaching is an economic, novel practice for extraction of metals from their sources by microorganisms. The current study aimed to extract uranium from Egyptian ores using native strains of actinomycetes. Two types of rocks and one ore sample were collected from west-central Sinai, Egypt. Major oxides of the samples and fourteen heavy metals, including uranium, were determined. X-ray diffraction analysis proved that uranium was present in the samples in various structures. Uranium was present in different concentrations, 220, 770, and 550 mg/kg in sandstone, granite, and manganese ore, respectively. Thirty-four actinomycete isolates were recovered from the studied samples using four different isolation media. Acid production capabilities were employed to select isolates for further leaching experiments. Bioleaching experiments were carried out using sterile and non-sterile ore samples. Using sterile ore samples, the highest solubilization percentages of U₃O₈ were 44.5, 38.55, and 16.76% from sandstone, manganese ore, and granite sample, achieved by isolates UA12, UA5, and U7, respectively. Lower solubilization percentages of U₃O₈ were recorded by using non-sterile ore samples. Investigating the factors affecting the bioleaching abilities of the tested organisms revealed that 10 days of incubation with 4% pulp density were the best conditions for U₃O₈ solubilization. The most efficient isolates were identified using 16S rRNA gene sequence analysis. UA12 identified to be Streptomyces bacillaris, while UA5 could not be identified, and U7 was assigned as uncultured bacterium clone. Scanning electron microscope examination of the bioleaching experiment showed different growth intensity within the active isolates. For larger-scale extraction purposes, a kilogram of sandstone, containing 220 mg of U₃O₈, was used in the form of a truncated cone in a heap leaching experiment. After 20 cycles, 14.72 mg/l (6.7%) of U₃O₈ was leached by S. bacillaris, while 19.36 mg/l (8.8%) of U₃O₈ was leached by chemical leaching using sulfuric acid. The results of this study prove that the extraction of uranium using actinomycetes could be exploited as less polluting, more economical, and more effective than traditional chemical extraction especially from low-grade ores or mining wastes.     

Laugung lateritischer Nickelerze mit heterotrophen Mikroorganismen

Organic acids that are excreted by microorganisms dissolve nickel from lateritic ores. In chemical leaching experiments, fifteen organic acids were tested in the concentration range of 0.05–0.5 M. The most effective were hydroxycarboxylic acids. The leaching of nickel is dependent on the type of mineralization. With completely limonitized ore, no mobilization occurred, while up to 90% of the nickel was extracted from silicate-bearing laterites by 0.5 M citric acid. In biological leaching tests, Penicillium was found to be the most effective microorganism. After improvement of the leaching conditions, up to 70% of the nickel was extracted at considerably lower citric acid concentrations than with the chemical leaching process. Generally, leaching of nickel from lateritic ores with heterotrophic microorganisms is possible and seems to be promising. Possibilities for future investigations are discussed.     

Decreased mineral availability enhances rock phosphate solubilization efficiency in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Microbial solubilization of rock phosphate (RP) is mainly achieved by the production of organic acids and medium acidification through H⁺ release. During RP solubilization, mineral nutrient availability is likely to be critical for determining how much carbon is channeled either for metabolite synthesis or for microbial growth, influencing organic acid release by microorganisms. Thus, the objective of this work was to study the relationships between the concentration of mineral nutrients in the growth medium and the efficiency of RP solubilization by Aspergillus niger FS1. For this, the fungus was grown in Czapek medium containing 0, 1, 2, 10, 50, and 100 % of its original concentration of mineral nutrients. Decreasing mineral availability in the growth medium led to decreases in fungal biomass and solubilized P, and increases in titratable acidity and solubilization efficiency as expressed by mg solubilized P per g fungal biomass (Y_P/B), indicating a shift in fungal metabolism from biomass production to organic acid release. The transfer of pre-grown biomass to media with or without added minerals confirmed that lower mineral availability increases Y_P/B and led to the solubilization of 76 % of P present in Patos RP. These observations open new perspectives on improving RP solubilization systems by manipulating mineral nutrient availability in the medium, with consequent gains in cost reduction.     

Biological leaching of sulfide minerals with the use of shake flask,aerated column,air-lift reactor,and percolation techniques

Four different experimental approaches were used to evaluate the microbiological leaching of ore material containing metal sulfides (Fe, Zn, Ni, Cu, Co) and aluminum silicates. A shake flask technique required the shortest contact time for the complete solubilization of the most readily leachable metals (Ni and Zn). Air-lift reactors and aerated column reactors required longer contact times and complete solubilization of either zinc or nickel was not achieved. The air-lift reactor approach was somewhat more effective than the aerated slurry technique. A percolation system was the least effective and yielded the lowest recoveries. Shake flasks (easily autoclavable) offered the advantage of comparison of the microbiological and solely chemical leaching. Aseptic conditions could not be maintained with the air-lift and aerated column reactors because of contamination via aerosol formation. In a relative scale the leaching patterns were similar in that the precipitation of Fe(III) occurred regardless of the technique; zinc and nickel sulfides were solubilized more quantitatively than those of copper and cobalt; aluminum concentrations, although high, indicated low leaching yields relative to aluminum silicates in the ore material; and the pH reached similar final values in the presence of bacteria.     

Leaching of copper ore of the Udokanskoe deposit at low temperatures by an association of acidophilic chemolithotrophic microorganisms

Pure cultures of indigenous microorganisms Acidithiobacillus ferrooxidans strain TFUd, Leptospirillum ferrooxidans strain LUd, and Sulfobacillus thermotolerans strain SUd have been isolated from the oxidation zone of sulfide copper ore of the Udokanskoe deposit. Regimes of bacterial-chemical leaching of ore have been studied over a temperature range from −10 to +20°C. Effects of pH, temperature, and the presence of microorganisms on the extraction of copper have been shown. Bacterial leaching has been detected only at positive values of temperature, and has been much more active at +20 than at +4°C. The process of leaching was more active when the ore contained more hydrophilic and oxidized minerals. The possibility of copper ore leaching of the Udokanskoe deposit using sulfuric acid with pH 0.4 at negative values of temperature and applying acidophilic chemolithotrophic microorganisms at positive values of temperature and low pH values was shown.     

Bio-based composite granules with simultaneous biocontrol and phosphorus fertilization roles: Outcomes from a lab-scale in vitro assessment

The use of phosphate rocks as low-solubility phosphorus fertilizers has been promoted to reduce the environmental impacts of agriculture, but adequate nutrient uptake by plants depends on solubilization of the rock, driven by soil microorganisms. Here, investigation was made of the microbial solubilization of low-solubility phosphate rocks, together with simultaneous bioprotective action involving the biocontrol of microorganisms. The aim was to enhance function and value by delivering two effects using a single bio-based product, in accordance with the concept of a “bioreactor-in-a-granule” system. A composite structure was developed, based on a starch matrix, comprising a combination of Trichoderma asperelloides, as a biocontrol agent, and Aspergillus niger, as an acidulant. A significant increase of up to 150% in P solubilization was achieved, indicating the positive effect of the microorganism-composite interaction. In vitro assays showed that the ability of T. asperelloides to inhibit Fusarium oxysporum mycelial growth was maintained in the presence of A. niger. Moreover, the estimated cost of the composite granule (0.35 US$/kg of product on a dry basis) revealed competitive. The results indicated that the association of T. asperelloides and A. niger is an effective way to increase nutrient availability and to inhibit plant pathogens, opening up possibilities for the design of multifunctional bio-based fertilizer composites.     

Non‐homogeneous biofilm modeling applied to bioleaching processes

A two‐dimensional non‐homogeneous biofilm model is proposed for the first time to study chemical and biochemical reactions at the microorganism scale applied to biological metal leaching from mineral ores. The spatial and temporal relation between these reactions, microorganism growth and the morphological changes of the biofilm caused by solid inorganic precipitate formation were studied using this model. The model considers diffusion limitations due to accumulation of inorganic particles over the mineral substratum, and allows the study of the effect of discrete phases on chemical and microbiological mineral solubilization. The particle‐based modeling strategy allowed representation of contact reactions between the microorganisms and the insoluble precipitates, such as those required for sulfur attack and solubilization. Time‐dependent simulations of chemical chalcopyrite leaching showed that chalcopyrite passivation occurs only when an impervious solid layer is formed on the mineral surface. This mineral layer hinders the diffusion of one kinetically determinant mineral‐attacking chemical species through a nearly irreversible chemical mechanism. Simulations with iron and sulfur oxidizing microorganisms revealed that chemolithoautotrophic biofilms are able to delay passivation onset by formation of corrosion pits and increase of the solid layer porosity through sulfur dissolution. The model results also show that the observed flat morphology of bioleaching biofilms is favored preferentially at low iron concentrations due to preferential growth at the biofilm edge on the surface of sulfur‐forming minerals. Flat biofilms can also be advantageous for chalcopyrite bioleaching because they tend to favor sulfur dissolution over iron oxidation. The adopted modeling strategy is of great interest for the numerical representation of heterogeneous biofilm systems including abiotic solid particles. Biotechnol. Bioeng. 2010;106: 660–676. © 2010 Wiley Periodicals, Inc.     

Microbial recycling of mineral waste products

The applicability of biotechnological methods to metal recovery from non-sulfide industrial waste products (slag, galvanic sludge, filter-press residue, filter dust, and fly ash) was investigated. From some products, copper, chromium, zinc or vanadium were completely extracted by sulfuric acid produced by Thiobacillus thiooxidans. The efficiency of bacterial metal solubilization varied depending on the type of waste material and on the pulp density. Stepwise increase of pulp density promoted bacterial growth and activity, resulting in metal concentrations of 6.6 g Cu/l, 6.3 g V/l, 24.4 g Zn/l or 21 g Cr/l in the leaching medium. In some cases bacterial leaching was as effective as chemical leaching with sulfuric acid. The efficiency of both processes is considered. In principal, bacterial metal recovery from industrial waste products seems to be feasible and may contribute to an increase in future supplies of raw materials, as well as to detoxification of industrial waste products resulting in reduced environmental pollution problems.     

Simultaneous sewage sludge digestion and metal leaching — effect of temperature

The effect of temperature on the simultaneous sewage sludge digestion and metal leaching process was studied in laboratory bioreactors of 20 l working volume. The results thus obtained showed that the process can be employed efficiently for metal solubilization, elimination of indicator microorganisms and sewage sludge stabilization at temperatures between 10°C and 30°C. Rates of pH reduction, sulfur oxidation, growth of thiobacilli, elimination of indicator microorganisms and solids degradation were found to decrease with temperature. Low metal solubilization efficiency was observed at 10°C; however, metals were solubilized to below the recommended level. The solubilization of organic matter and nutritive elements (N, P and K) was not significantly affected by the variation in temperature. The fertilizer value of sludge after leaching and digestion did not change significantly and remained the same irrespective of temperature. *** DIRECT SUPPORT *** AG903078 00005     

Isolation and Characterization of Coal Solubilizing Aerobic Microorganisms from Salt Range Coal Mines,Pakistan

Microbial solubilization of coal has been considered as a promising technology to convert raw coal into valuable products. In the present study, initially a total of 50 different aerobic bacterial and fungal isolates have been isolated from soil, coal and water samples of Dulmial Coal Mines, Chakwal, Pakistan, but on the basis of solubilization potential, only four isolates were selected for further study. The intensity of biosolubilization was measured by determining the weight loss of the coal pieces, which was observed to be about 25.93% by Pseudomonas sp. AY2, 36.36% by Bacillus sp. AY3 and 50% by Trichoderma sp. AY6, while Phanerochaete sp. AY5 showed maximum coal solubilization potential i.e. 66.67% in 30 days. UV/Vis spectrum revealed an increase in the pattern of absorbance of all treated samples compared to control referring to solubilization. Fourier transform infrared spectroscopy indicated alterations in the structure of treated coal in comparison to control coal suggesting breakdown in the complex structure of coal. The major absorbance bands in infrared spectroscopy for solubilization product were attributed to carbonyl (1,600 cm^?1), hydroxyl (3,450 cm^?1), cyclane (2,925 cm^?1), ether linkage (1,000–1,300 cm^?1), carboxyl (3,300–2,500 cm^?1) and side chains of aromatic ring (1,000–500 cm^?1). The presence of microorganisms and surface erosion of coal residues compared to control samples were observed by scanning electron microscopy, which suggested that isolated microorganisms were able to survive in coal for a longer period of time. Therefore, the present study concluded that microorganisms isolated from coal mines have excellent potential for coal solubilization which is considered as a crucial step in coal methanogenesis allowing them to be used successfully for in situ methane production to meet future energy demands.     

Leaching of iron from kaolins by a spent fermentation liquor: Influence of temperature,pH, agitation and citric acid concentration

Summary The efficiency of a biological bleaching process on a highly contaminated iron oxides kaolin was analyzed. The bio-bleaching method consists of two steps: first, the fermentation of a chemically-defined medium byAspergillus niger leads to a spent liquor which, in a second step, is employed as leaching agent for kaolin. In the leaching process 43% of iron oxides was removed when the kaolin was treated at 60°C for 5 h and the whiteness index was increased to 67%. Extracellular enzymes were not responsible for leaching, which is basically due to organic acids produced during the fermentation.     

Effect of Cell Lysis (CLs) Products on Acidophilic Chemolithotrophic Microorganisms and the Role of Acidocella Species

Acidophilic chemolithotrophic microorganisms (CMs) are widely used for bioleaching of mineral resources. However, the growth of bacteria and their leaching activity are often inhibited (restricted) by organic components, e.g. lysates and exudates. The aims of this study were to examine the extent of cell lysis (CLs) inhibition on acidophilic microorganisms and to identify microorganisms that can utilize CLs products and eliminate their inhibition effect on acidophilic microorganisms. Specifically, it was revealed that Acidithiobacillus caldus was severely inhibited at 5% CLs products, whereas A. ferrooxidans and Leptospirillum ferriphilum are severely inhibited at 20%. It has been found that strains RBA and RBB of heterotrophic bacteria, isolated from anaerobic sludge, can biodegrade CLs products and when co-cultured with A. ferrooxidans, they can alleviate the toxic effect of CLs products under low pH (2–3). It has been shown that besides CLs, isolated strains can grow on glucose, glycerol, yeast extract, citric acid, and tryptone soya broth with an optimum temperature of 35°C and a pH of 3. The strains showed the ability to reduce ferric ions to ferrous ions when glycerol was used as a substrate after 2 days under both aerobic and anaerobic conditions. On the basis of morphophysiological and molecular biological studies, the isolated strains RBA and RBB were identified as Acidocella spp.     

Use of heterotrophic microorganisms in mineral biotechnology

A process for biological removal of iron from quartz sands, kaolins and clays was developed in which these industrial minerals were leached at 90°C with lixiviant produced as a result of the cultivation of acid-producing heterotrophic microorganisms, mainly strains of Aspergillus niger, at 30°C in a nutrient medium containing molasses as a source of carbon and energy. The lixiviant, i.e. the fermentation fluid, contained oxalic and citric acids as main components and after the cultivation was acidified to a pH of 0.5 by means of hydrochloric acid. The leaching was carried out in mechanically stirred acid-resistant vats for a period of from 1 to 5 hours. The iron content of some sands treated by this method was lowered from 0.035–0.088 to below 0.012% Fe₂O₃ making them suitable for the preparation of high quality glass. The iron content of different kaolins was lowered from 0.65–1.49 to 0.44–0.75% Fe₂O₃ and as a result of this their whiteness was increased from 55–87 to 86–92%. The iron content of a clay was lowered from 6.25 to 1.85% Fe₂O₃ and this increased the fireproofness of the clay from 1 670 to 1 750°C. Similar process was used for leaching of aluminium from aluminosilicates, mainly clays and kaolins. However, after the cultivation the fermentation fluid was acidified either by means of sulfuric or hydrochloric acid or by means of different mixtures of inorganic acids. For enhancing aluminium solubilization the aluminosilicates were heated before leaching at 600–650°C for 1–2 hours. Over 90% of the aluminium present in different clays and kaolins was leached within 3–6 hours in this way. “Silicate” bacteria related to the species Bacillus circulans and B. mucilaginosus were used to leach silicon from low-grade bauxite ores containing aluminosilicates as impurities. The bacterial action was connected with the formation of mucilaginous capsules consisting of expolysaccharides. The solid residues after leaching were characterized by higher values of alumina content and were suitable for processing by means of the BAYER process for recovering aluminium. Heterotrophic bacteria were used to leach manganese from oxide ores using different organic compounds as reducing agents.     

Simultaneous phosphate solubilization potential and antifungal activity of new fluorescent pseudomonad strains, Pseudomonasaeruginosa, P. plecoglossicida and P. mosselii

Of 80 fluorescent pseudomonad strains screened for phosphate solubilization, three strains (BFPB9, FP12 and FP13) showed the ability to solubilize tri-calcium phosphate (Ca₃(PO₄)₂). During mineral phosphate solubilization, decrease of pH in the culture medium due to the production of organic acids by the strains was observed. These phosphate solubilizing strains produced indole-3-acetic acid (IAA) and protease as well as exhibited a broad-spectrum antifungal activity against phytopathogenic fungi. When tested in PCR using the gene-specific primers, strain BFPB9 showed the presence of hcnBC genes that encode hydrogen cyanide. On the basis of phenotypic traits, 16S rRNA sequence homology and subsequent phylogenetic analysis, strains BFPB9, FP12 and FP13 were designated as Pseudomonas aeruginosa, P. plecoglossicida and P. mosselii, respectively. Present investigation reports the phosphate solubilization potential and biocontrol ability of new strains that belong to P. plecoglossicida and P. mosselii. Because of the innate potential of phosphate solubilization, production of siderophore, IAA, protease, cellulase and HCN strains reported in this study can be used as biofertilizers as well as biocontrol agents.     

The Amount of Phosphate Solubilization Depends on the Strain,C-Source,Organic Acids and Type of Phosphate

Although production of organic acids (OAs) is usually mentioned as the main mechanism of phosphate solubilization, the relationship between carbon sources (C-sources) and OAs produced during phosphate-solubilization by microorganisms is still poorly understood. We evaluated the influence of different C-sources on FePO₄·2H₂O and Ca₃(PO₄)₂ solubilization by bacteria and on the identity/quantity of the OAs produced. Our results showed that the amount of phosphate solubilization depends on the strain, C-source, OAs, and type of phosphate. Among the five strains under study isolated from cowpea nodules (Rhizobium tropici strain UFLA 03-08, Acinetobacter sp. strain UFLA 03-09, Paenibacillus kribbensis strain UFLA 03-10, P. kribbensis strain UFLA 03-106, and Paenibacillus sp. strain UFLA 03-116), three of them solubilized Ca₃(PO₄)₂ in all C-sources. The influence of C-sources on Ca₃(PO₄)₂-solubilization increased in the following order: cellulose?<?lactose?<?mannitol?<?glucose. A significant positive correlation between the amount of phosphorus solubilized from Ca₃(PO₄)₂ and the concentration of total OAs in the presence of glucose and mannitol was observed for these three strains. In the presence of glucose, the highest solubilization rates are associated with high concentrations of tartaric acid, and in the presence of mannitol, are associated with maleic acid. Only one strain produced OAs in the medium with lactose and Ca₃(PO₄)₂, but there was no OAs in the medium containing cellulose. Despite the production of OAs, albeit in small concentrations, in all the C-sources investigated, FePO₄·2H₂O-solubilization was not observed. Thus, a relationship among C-sources, OAs, and phosphate solubilization was not always verified.     

Effects of Mineral Structure and Microenvironment on K Release from Potassium Aluminosilicate Minerals by Cenococcum geophilum fr

K-bearing minerals with enormous reserve in the world, were not easy to provide the available K nutrient element for plant direct uptake because of the slow K release rate. With the potential potassium-solubilizing microorganisms, the slow K release rate from minerals would be improved significantly. In this work, Cenococcum geophilum Fr, one of the most common ectomycorrhizal fungi in boreal to temperate regions, was adopted to dissolve K-bearing minerals for K release. Five kinds of potassium aluminosilicate minerals were tested by bioleaching experiments in pure culture, including feldspar, nepheline, biotite, muscovite and illite. The available and unavailable potassium amounts in minerals before and after bioleaching were measured and compared with each other. The effect of mineral structure on the potassium solubilization efficiency by Cenococcum geophilum Fr. was discussed. Furthermore, the microenvironment formation between fungi and mineral surface to enhance the K release rate was investigated through detecting K, Al, Si concentrations and metabolites amounts (polysaccharide and organic acids) in microenvironment and external environment, respectively. Experimental results demonstrated that Cenococcum geophilum Fr. was a potential candidate of potassium solubilizing microorganisms, and both mineral structure and microenvironment have significant effects on the K release rate.     

Microbial community study of the iron ore concentrate of the Sishen Iron Ore Mine, South Africa

As a result of the advancing global technologies and civilisation, there has been a progressive depletion of high-grade mineral deposits. Consequently, it has become increasingly important to process lower-grade ores. Phosphorous (P) and particular potassium (K) contained in the iron ore concentrates of the Sishen Iron Ore Mine have a detrimental effect on the steel making process, whereby these alkali’s cause cracks to form in the refractory lining of blast furnaces. It is initially essential to determine which microbes are indigenously present at the Sishen Iron Ore Mine before strategising how best to employ them to industrial advantage. Therefore, the objective of this study was to determine which microorganisms are indigenous to the iron ore and soil of the Sishen Iron Ore Mine. The bacterial 16S PCR and fungal ITS PCR revealed several bacterial and fungal species present in the mine environment. The bacterial isolates were found to be closely related to Herbaspirillum species, as well as Acidithiobacillus ferrooxidans, while the fungal isolates were closely related to Aureobasidium pullulans, Phaeosphaeria nodorum, Aspergillus fumigatus, and Candida parapsilosis. Isolating A. fumigatus from the iron ore/soil of the mine may indicate that A. niger, the most common fungi used for the production of citric acid, can adapt to the stringent mine environment. This would allow the application of A. niger for the production of citric acid, which may be used for the chemical leaching of the P and K from the iron ore concentrate of the Sishen Iron Ore Mine.     

Copper removal from an industrial waste by bioleaching

Summary Copper contained in a solid industrial waste produced in a silicone manufacturing process was leached with spent iron medium from aThiobacillus ferrooxidans culture. Most effective leaching was observed in a continuously fed, dual reactor system. Spent iron medium was generated by growingT. ferrooxidans in 0.9 K iron medium at pH 1.5 in the first reactor, and was transferred to a second reactor in which it leached the copper from the waste. Leaching was effective at a pulp density of the waste material as high as 20%. In experiments run at a pulp density of 2.5%, the spent iron medium was most efficient in leaching copper when it was first diluted 100-fold with a mineral salts solution at pH 1.5. Removal of the copper from the waste appeared to involve its displacement by acid, dissolved mineral salts, and ferric iron. Potentials for practical application of this process are discussed.     

Leaching of copper converter slag withAspergillus niger culture filtrate

Leaching of copper converter slag of M/s Hindustan Copper Ltd, Ghatshila (Bihar, India) was carried out usingAspergillus niger culture filtrate. The effects of the duration of leaching, temperature, pulp density and the addition of hydrochloric acid were studied.A. niger culture filtrate solubilized metals from the converter slag at levels of 18.70% copper, 7.40% nickel and 4.00% cobalt. Addition of hydrochloric acid was found to improve copper, nickel and cobalt solubilization to 46.52, 27.90 and 37.96%, respectively. HPLC analysis of the fungal culture filtrate revealed the presence of succinic and citric acids. Therefore, leaching of the slag was also carried out with matching concentrations of these organic acids individually as well as with both mixed together. Results are discussed.