- Email: [email protected]
Effect of mineral constituents in the bioleaching of uranium from uraniferous sedimentary rock samples, Southwestern Sinai, Egypt
Journal of Environmental Radioactivity 134 (2014) 76e82
Contents lists available at ScienceDirect
Journal of Environmental Radioactivity journal homepage: www.elsevier.com/locate/jenvrad
Effect of mineral constituents in the bioleaching of uranium from uraniferous sedimentary rock samples, Southwestern Sinai, Egypt Maisa M. Amin a, Ibrahim E. Elaassy a, Mohamed G. El-Feky a, Abdel Sattar M. Sallam b, Mona S. Talaat b, Nilly A. Kawady a, * a b
Nuclear Materials Authority, Cairo, Egypt Faculty of Science Ain Shams University, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 July 2013 Received in revised form 18 February 2014 Accepted 26 February 2014 Available online 28 March 2014
Bioleaching, like Biotechnology uses microorganisms to extract metals from their ore materials, whereas microbial activity has an appreciable effect on the dissolution of toxic metals and radionuclides. Bioleaching of uranium was carried out with isolated fungi from uraniferous sedimentary rocks from Southwestern Sinai, Egypt. Eight fungal species were isolated from different grades of uraniferous samples. The bio-dissolution experiments showed that Aspergillus niger and Aspergillus terreus exhibited the highest leaching efficiencies of uranium from the studied samples. Through monitoring the biodissolution process, the uranium grade and mineralogic constituents of the ore material proved to play an important role in the bioleaching process. The tested samples asserted that the optimum conditions of uranium leaching are: 7 days incubation time, 3% pulp density, 30 C incubation temperature and pH 3. Both fungi produced the organic acids, namely; oxalic, acetic, citric, formic, malonic, galic and ascorbic in the culture filtrate, indicating an important role in the bioleaching processes. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Uranium bioleaching Uraniferous sedimentary rock Aspergillus niger Aspergillus terreus Fungal organic acids
1. Introduction Bioleaching is a specialized biohydrometallurgical process principally based on the ability of microorganisms to transform solid compounds into soluble and extractable elements. The extracted elements could be removed through several techniques (Akcil and Deveci, 2010; Ehrlich, 2004). Metal prices, are not the only motivating factor for employing the biohydrometallurgical process (Brierley, 2008), cost production (Gericke et al., 2009; Moore, 2008) and difficulty in recognition as well as evaluation of ore deposits by conventional processes are among other factors (Brierley, 2010). Fungal leaching of metals from minerals is mainly based upon three synchronous steps. Acidolysis which protonation of oxygen atoms that occurred around the surface of metallic compounds. The proton and oxygen associated with water displace the metal from the surface (Johnson, 2006; Mulligan et al., 2004; Xu and Ting, 2009). Complexolysis, where metal complex formation results in the solubilization of the metal ion e.g. complex of oxalic acid and
* Corresponding author. Tel.: þ20 (0)1008123944. E-mail address: [email protected] (N.A. Kawady). http://dx.doi.org/10.1016/j.jenvrad.2014.02.024 0265-931X/Ó 2014 Elsevier Ltd. All rights reserved.
iron. Furthermore, this process often reduces the toxicity of heavy metals towards fungi (Johnson, 2006). Redoxolysis, when reduction of metal ions occurs in an acidic environment, such as in the case of ferric ions and/or manganese under the influence of oxalic acid (Bromacher et al., 1997). A series of organic acids is formed by fungal metabolism, resulting in acidolysis complex and chelate formation (Mulligan et al., 2004). Lee et al. (2005) described the effect of several factors on uranium bioleaching efficiency of uranium bearing black shale (349 mg kg1 of uranium) using Acidothiobacillus ferrooxidans (iron-oxidizer organism). They observed that the initial inoculations of bacterial cells of batch-type reactors, containing black shale, caused lowering of pH and highering of redox potential as well as of Fe3þ in the aqueous leach solutions over the non-inoculated reactor up to 200 h. Ores are classified into high grade (where metal concentration is relatively high) and low grade (with low concentration of metals, like shale) (Chow et al., 2010). Shales are sedimentary rocks formed during the latter part of the Cambrian period and up to the first part of the Ordovician period, approximately 540e 480 Ma ago (Falk et al., 2006). Their color is commonly tones of gray, brown, green or black due to the presence of some organic pigments in microbes inhibiting the shales. The introduction of quartz causes an increase in the size of their grains and transforms
M.M. Amin et al. / Journal of Environmental Radioactivity 134 (2014) 76e82
them into sandstones. Shale deposits containing the elements Fe, K, Si and Al are most susceptible to microbial transformation (Harris, 2005; Piper and Calvert, 2009). The lower Carboniferous (upper Visean) Um Bogma Formation (Fig. 1) in the southern extreme of Wadi (Vally) Naseib consists of three members, namely lower sandy dolostone, middle siltstone marl intercalations and upper dolostone (Kora, 1984). Normally Karstification process affects the lower (sub-soil) and middle (top soil) to form another soil profile hosting several metallic forms (El Sharkawi et al., 1990). El Aassy et al. (2000) pointed out that lateritization processes affected all three members. These three members are the main target of this study. The aim of this work focused upon using the different biomasses (fungi) isolated from three sedimentary samples (natural habitat) in the bioleaching of uranium from these samples.
77
2. Materials and methods 2.1. Collection of ore samples To represent the Allouga mountain, in southern Wadi Nasieb, three radioactive geologically grapped samples were collected in sterile polyethylene bags from the prospective area in southwestern Sinai, Egypt. These samples (Table 1) were chosen to represent different grades of uranium concentrations. 2.2. Mineralogical and chemical analyses of studied samples An X-ray diffraction technique was used to identify the mineralogical constituent of the study sample using (PHILIPS PW 3710/ 31) diffractometer with automatic sample changer PW 1775, (21
Fig. 1. Geological map of the studied localities, southwestern Sinai, Egypt (After, Alshami, 2003).
78
M.M. Amin et al. / Journal of Environmental Radioactivity 134 (2014) 76e82
at the Regional Center for Mycology and Biotechnology, Al Azhar University, Cairo, Egypt.
Table 1 Samples collected from the three members of Um Bogma Formation. A-1
Sandy dolostone, grey to brown, medium hard, with Cu mineralization (lower member). Grey shale with sulfur patches (upper member). Sandy dolostone with shale interlayer, grey to brown shale (middle member).
A-2 A-3
positions), Scintillation counter, Cu-target tube and Ni filter at 40 kV and 30Am. This instrument is connected to a computer system using X-40 diffraction program and ASTM cards for mineral identification. The pulverized samples were analyzed by a conventional wet chemical technique (Shapiro and Brannock, 1962). The SiO2, Al2O3, TiO2 and P2O5 were constituents recognized using a spectrophotometric method. The contents of Na and K were determined using a flame photometric technique. On the other hand, the total oxides as Fe2O3, MgO and CaO were evaluated by titration methods. The loss on ignition (L.O.I) was calculated gravimetrically. The estimated error for the major constituents is less than 1%. Uranium was analyzed by titration against NH4VO3 (Daveis and Gray, 1964). All the chemical analyses were carried out in the laboratories of the Nuclear Materials Authority, Egypt. 2.3. Microbiological studies The microbial studies include the fungal isolation from the study rock samples and the conditioning of the bioleaching factors as follows: 2.3.1. Media and culture conditions used in bioleaching The media used in this work are Dox agar medium of composition g/L: NaNO3, 2 (0.0325); KH2 PO4, 1 (0.00735); MgSO4$7H2O, 0.5 (0.002033); KCl, 0.5 (0.00676); FeSO4$5H2O trace; sucrose, 30 (0.0877); agar, 15.5 g and yeast extract added to initiate fungal growth. The pH value of the media was adjusted to be 6.5 before autoclaving at 120 C and 1.5 atm. for 20 min. Two techniques were used in fungal isolation from the ore. The first one is the directplating technique, in which fine ore-powder was spread directly on the surface of Dox agar plates under aseptic conditions. The agar plates were incubated at 28 C 2 until the fungal colonies clearly appeared. The second technique is the dilution plating in which 1 g of the ore powder was taken under aseptic conditions and mixed well with 9 ml of sterile distilled water; 0.1 ml of this mixture was spread under aseptic conditions by sterile glass rod on the surface of an agar plate. The plates were incubated at 28 C 2 until development of the colony. The hyphal tips of each colony were removed and plated on the surface of the agar plates. The developed colonies were examined with a microscope to detect contamination. The pure isolated fungi were identified according to Gilman (1957) and Pitt (1979). 2.3.2. Determination of organic acids produced by Aspergillus niger and Aspergillus terreus Both tested organisms were cultivated on Dox liquid media for 7 days at 30 C. Organic acids were recognized in the filtrates by HPLC
2.3.3. Factors affecting the bioleaching of uranium Several factors were investigated in the aim of obtaining the optimum conditions for solubilization of uranium. These factors are fungal activity, pulp densities on fungal growth, incubation periods, incubation temperatures and initial pH values. One hundred ml of Dox liquid medium was placed in 250 ml Erlenmeyer flasks. The flasks supplemented with ore samples concentration. Triplicate sets of flasks were prepared for each organism and ore. The flasks were autoclaved at 1.5 atm for 20 min, after cooling the flasks were inoculated with 1 ml spore suspension. At the end of the incubation period, the culture medium was filtered and centrifuged at 4000 rpm to precipitate more particles, repeated for each factor. The filtrate was kept for uranium determination. 3. Results and discussion 3.1. Mineralogical and chemical analyses of studied samples The mineralogical composition of studied ore samples showed that A-1 consisted mainly of dolomite and gypsum, A-2 consisted of kaolinite and quartz and A-3 consisted of dolomite with ferron dolomite and quartz. The residual mineralogy of the sample A-3 which exhibited high bioleaching efficiency, showed that the rest of the dolomite complexes with the iron (in the original sample) to form ankerite [Ca (MgO.67 FeO.33) (CO3)2]. The chemical analysis of the studied uraniferous sedimentary rocks, which are used in this study, revealed the presence of high contents of SiO2 and Al2O3 in sample A-2 and high contents of CaO, MgO as well as loss on ignition (L.O.I) in samples A-1 and A-3 (Table 2). From this table there are three uranium categories; high grade (A-1) with uranium concentration 800 ppm, moderate grade (A-2) with 660 ppm uranium concentration and (A-3) of the lowest grade uranium concentration 77 ppm. 3.2. Fungal activity on uranium bioleaching An obvious variation in the leaching efficiencies appears with the different investigated fungal species (Table 3) in the following order: Aspergillus species > Penicillium species > Mucor species. Meanwhile, within Aspergillus species the A. niger displayed the highest uranium leachability whereas in Penicillium species, the P. diversum revealed the highest uranium leachability. Variations in bioleaching efficiencies of uranium are strongly influenced by the ore mineralogy, the metal loss through electro-sorption properties of the ore as well as effective and selective leaching by certain strains for some heavy metals more than others (Valix et al., 2001). The optimization parameters affecting bioleaching process were determined upon applying the testes of highly ability fungal strains (A. niger and A. terreus) to uranium solubilization. 3.3. Pulp density and fungal growth inhibition Growth of all Aspergillus species, Penicillium species and Mucor species were noticeably decreased with increasing the pulp density
Table 2 The chemical analyses of the tested samples (wt. %). Sample
SiO2
Al2O3
Fe2O3
MnO
CaO
MgO
Na2O
K2O
P2O5
TiO2
L.O.I
Total
U
A-1 A-2 A-3
17.7 78.6 11.4
4.59 8.67 3.06
0.044 0.032 0.03
0.481 0.02 0.43
22.4 2.24 26.9
15.7 1.6 16.9
0.236 0.18 0.307
0.539 1.173 0.337
0.161 0.615 0.142
0.19 0.53 0.111
37.9 6.32 40.6
99.94 99.99 100.21
0.08 0.066 0.0077
M.M. Amin et al. / Journal of Environmental Radioactivity 134 (2014) 76e82 Table 3 Effect of different ore concentrations (A-1, A-2 and A-3) on the inhibition efficiency (%) of isolated fungi. Fungus sp.
A. niger
A. terreus
A. Fumigatus
A. flavus
P. steckii
P. italicum
P. diversum
Mucor sp.
Sample no.
A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3
Table 4 Bioleaching efficiency of uranium by using the dominant fungal isolates. Fungus species
Sample
Uranium leaching efficiency (%)
Final pH
A. niger
A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3
34 51 57 28 36 42 20 23 33 18 25 28 13 18 21 12 20 25 15 27 33 8 14 22
5.64 5.43 5.38 5.52 5.18 4.74 5.97 6.06 5.63 5.77 6.36 5.43 5.65 5.68 5.11 6.47 6.64 6.81 4.87 5.34 5.65 6.66 6.53 6.21
Inhibition efficiency (%) 1%
2%
4%
6%
8%
16.9 3.1 12.1 10.2 14.6 25.2 1.4 4.3 14.0 4.7 12.1 26.3 0.4 0 15.6 1.3 1.2 5.2 2.7 6.7 16.5 12.0 13.3 12.0
26.3 13.2 32.9 24.7 22.4 28.6 16.3 16.6 15.5 3.3 9.1 28.5 1.5 2.1 20.1 9.9 2.3 11.4 13.9 29.8 21.0 24.0 25.6 33.5
40.4 40.4 53.4 27.7 45.6 44.3 31.4 41.4 44.6 32.5 45.9 59.6 2.6 41.5 30.3 11.2 24.5 27.4 16.4 42.0 45.0 38.9 35.0 44.7
67.6 60.2 65.4 58.5 53.4 57.8 54.8 71.0 58.5 54.0 42.4 61.2 29.2 45.6 37.9 19.6 29.4 38.7 29.9 45.9 51.1 59.5 61.5 69.9
77.1 72.3 77.0 71.9 78.8 78.6 81.6 86.1 67.9 79.9 72.9 71.0 37.4 51.8 41.5 30.2 42.2 41.4 76.2 64.9 68.8 81.3 77.3 73.5
except A. flavus at 2% in both A-1 and A-2 and P. italicum for A-2. After 4% pulp density, the growth of Penicillium species slightly decreased with increasing of the pulp density in the media. This observation suggested that Penicillium species have an ability to grow under stress of uranium concentration more than Aspergillus species. In addition, growth inhibition may be due to increase in the concentration of heavy metals in the leaching environment leading to increase in toxicity, which might have inhibited the growth of the microorganisms (Xu and Ting, 2004). Growth of all isolated fungal strains substantially highly decreased with increasing the concentrations of the used ore samples except P. steckii, P. italicum and P. diversum. This trend changed in the sample A-1 at concentration 4% which led to percentage of growth inhibition that reached 13.8%, 2.5%, 11.2% and 16.4% respectively (Tables 3 and 4). This may be attributed to the ability of this organism to tolerate and grow under stress of uranium. The 1% pulp density apparently proved to be the best for all fungal growth between all concentrations. The highest uranium concentration sample (800 ppm) showed the highest growth in the 1% concentration in A. terrus, A. fumigatus, A. flavus, P. diversum and Mucor sp., while the others A. niger, P. steckii and P. italicum gave high growth in the moderate uranium concentrations (660 ppm). This indicates that the uranium concentrations as well as the lithologic type play an appreciable role in the growth of the fungal species. 3.4. Bioleaching controlling factors The fungal strains of high bioleaching efficiency (A. niger and A. terreus) were selected to determine their bioleaching controlling factors as follows: 3.4.1. Pulp densities Leaching of uranium by fungal strains A. niger and A. terreus increased upon increasing the pulp densities in the growth media up to 3% in the three different grades of uranium ore samples, A-1, A-2 and A-3. The highest percentage of uranium solubilization, on the other hand, resulted from the ore sample with the lowest grade
79
A. terreus
A. fumigatus
A. flavus
P. steckii
P. italicum
P. diversumum
Mucor sp.
of uranium concentration and having ferrous dolomite, as shown in Fig. 2. The best leaching efficiency of uranium using fungal strains occurred at 3% of sample A-3, which reached 80% by A. niger and 56% by A. terreus. At a value 3% pulp density, appears that the leaching efficiency of uranium decreased upon increasing the pulp densities. This may be due to insufficient oxygen transfer, limited nutrition provided to the microbes or deformation of cells in the high solid concentrations, as described by Mohapatra et al. (2007). The decrease in the bioleaching efficiency by increasing the sample concentrations may be due to the final pH value which was found to increase with increasing the sample concentrations (but still less than the initial pH of the medium). This led to an increase in the binding capacity of the fungal mycelium at the higher concentrations, as mentioned by Hefnawy et al. (2002). Meanwhile, this led to the decrease of metal concentrations in the actual leaching liquor and the decrease of the solubilization efficiency, as investigated by Tang and Valix (2006). Santhiya and Ting (2005), Aung and Ting (2005) and Liu et al. (2007) mentioned that the optimum pulp density in the bioleaching was 1% (w/v). On the other hand, Ibrahim et al. (2007) obtained the maximum solubilization of uranium and alumina at 4% (w/v) from gibbsite, dolostone and mudstone ores by A. niger. 3.4.2. Incubation periods Uranium leaching by A. niger was highly affected by the incubation periods. The best solubilization activity occurred upon 7 days of incubation; where the amounts of uranium, solubilized from the samples A-1, A-2 and A-3, were 45.7%, 56% and 64% respectively. However, after 7 days of incubation the amounts of uranium solubilized slightly decreased with increasing incubation periods up to 10 days. The same pattern of solubilization of uranium by A. niger was followed by A. terreus and resulted in 27%, 35% and 47% leaching efficiency for A-1, A-2 and A-3, respectively (Fig. 3). The amount of uranium solubilized in the growth media slightly decreased with increasing the incubation period above 7 days. These results were in agreement with those obtained by El Sayed (2012), where eight and six days were the optimum incubation periods for A. niger and A. fumigatus to solubilize uranium and REEs from gibbsite-bearing shale sample.
80
M.M. Amin et al. / Journal of Environmental Radioactivity 134 (2014) 76e82
Fig. 2. Relation between different Pulp densities and Uranium Solubilization by A. niger and A. terreus at 30 C for 7 days: (a) A-1, (b) A-2 and (c) A-3.
Fig. 3. Relation between different Incubation Periods and Uranium Solubilization by A. niger and A. terreus at 30 C: (a): A-1, (b): A-2 and (c): A-3.
3.4.3. Incubation temperatures Uranium leaching from its ores was found to be highly affected by the incubation temperature; it reached its maximum at 30 C. At this temperature, A. niger solubilized 43%, 54% and 64% of uranium found in the ore samples A-1, A-2 and A-3 respectively (Fig. 4a, b and c). Whereas, uranium leached by A. terreus at 30 C revealed 26%, 34.8% and 45% solubilization respectively. At this incubation temperature, the final pH value of the medium shifted toward acidity. El Sayed (2012) mentioned that the maximum bioleaching of heavy metals occurred at 30 C for A. niger and 35 C for A. fumigatus. At higher temperatures (40 C and 45 C), though, the metabolic rate of organism decreases and the organism might even not survive (Mohapatra et al., 2007).
pH 3 is apparently the best for bioleaching efficiency of uranium. It reached 65% and 49.3% for the 1% sample A-3 by A. niger and A. terreus respectively. Moreover, the increasing of the growth media pH value led to an increase in the binding capacity of the fungal cells as well as a decreasing the leaching efficiency of uranium (Hefnawy et al., 2003), yet still less than the initial pH value of the medium. Amin (2007) suggested that the maximum solubilization of uranium was obtained at the initial pH value of 4. The variation in the optimum initial pH value of metal solubilization may be due to the different chemistry of the metal complexation, as previously described by Macaskie and Dean (1989); Yan and Viraraghavan (2003). The pH value of the medium is very important for the growth of microorganisms, for the character of their metabolism and hence for the biosynthesis of their antimicrobial products due to secondary metabolites (Patterson and Boils, 1995).
3.4.4. Initial pH The optimum pH value was determined by using the fungal strains which have the highest leachability at 1% from samples A-1, A-2, and A-3 at 30 C for 7 days on Dox-liquid media. Fig. 5 showed that the amount of released uranium decreased with increasing the initial pH value of the growth media. The highest bioleaching efficiency of uranium from the samples was obtained at pH 3 by A. niger followed by A. terreus. The final pHs increased with increasing initial pH values of the growth medium. The optimum
3.5. Role of organic acids and mineral constituents The application of bioleaching technology to uranium leaching results in an additional acid generation as a consequence of microbial activity. Thus leads to a decrease of acid consumption at uranium industrial plants (Umanskii and Klyushnikov, 2013).
M.M. Amin et al. / Journal of Environmental Radioactivity 134 (2014) 76e82
81
Fig. 5. Relation between different Initial pHs and Uranium Solubilization by A. niger and A. terreus for 7 days: (a) A-1, (b) A-2 and (c) A-3. Fig. 4. Relation between different Incubation Temperatures and Uranium Solubilization by A. niger and A. terreus for 7 days: (a): A-1, (b): A-2 and (c): A-3.
Acidolysis is the principal mechanism in bioleaching of metals by microbes which produces organic acids such as citric, oxalic, gluconic and malic during bioleaching processes (Johnson, 2006). Organic acids dissolve metals from minerals by displacement of metal chelates (Ren et al., 2009). Accordingly, different mineral constituents of tested ore samples with different binding chelation need different types and concentrations of produced organic acids. This, on the other hand, played a direct and important role in the bioleaching process, and confirms that depletion of bioleaching agents could be one reason in decreasing the rate of metal extraction (Amiri et al., 2012). There are two important functions in bioleaching. First, they can facilitate the dissolution of metal ions from leaching materials through chelation of the metals released in solution and also destabilize the bonds between the surface metal and bulk leaching materials (Gräfe et al., 2011). Second, they can reduce the adverse effects of metal ions imposed on microorganisms through chelation or complexation (Burgstaller and Schinner, 1993). Metabolites of both fungal strains were subjected to proper chromatographic analysis (Fig. 6a and b). The analysis proved that A. niger secretes, in the growth media, organic acids such as, formic, acetic, citric and oxalic. This agrees to great extent with previous,
A. niger generating organic acids like oxalic, citric, malic and tartaric, which are effective for metal solubilization (Anjum et al., 2009; Mulligan et al., 2004; Murad et al., 2003). Whereas, A. terreus secreted citric, oxalic, galic, malonic and ascorbic as secondary metabolites, which were effective in uranium solubility. The organic acids produced by A. niger and A. terreus under 1% and 5% bioleaching processes are shown in Fig. 6. These acids are essential for the leaching of heavy metals from ore materials and solid wastes (Ren et al., 2009; Santhiya and Ting, 2005). Oxalic acid is the major lixiviate among the metabolites product by A. niger, which yielded highest extraction of metals at 1% pulp density in 60 days (Santhiya and Ting, 2005). These dissoluted metals were related to different mineral constituents of the ore samples. 4. Conclusions The following conclusions were drawn from this study: 1 The eight fungal species were identified as A. niger, A. terreus, A. fumigatus, A. flavus, P. italicum, P. diversum, P. steckii and Mucor species. A. niger and A. terreus were the only isolates which achieved the highest leaching efficiency of uranium from the studied uraniferous samples.
82
M.M. Amin et al. / Journal of Environmental Radioactivity 134 (2014) 76e82
Fig. 6. Typical organic acids chromatograph in fungi grown on Dox liquid medium (a) A. niger (1: formic acid; 2: oxalic acid; 3: citric acid and 4: acetic acid) and. (b) A. terreus, (1: oxalic acid; 2: citric acid; 3: ascorbic acid; 4: malonic acid; and 5: galic acid).
2 All tested fungi showed variations in bioleaching directly related to variations in mineral constituents and uranium grades. 3 The optimum conditions of uranium leaching from its ores, using A. niger and A. terreus, were found to be 7 days incubation time, 3% pulp density pH 3 and 30 C incubation temperature. 4 A. niger and A. terreus exhibit an appreciable potential in generating a variety of organic acids which play an important and effective role for uranium leaching. References Akcil, A., Deveci, H., 2010. Mineral biotechnology of sulphides (Chapter 4). In: Jains, S., Khan, A., Rai, M.K. (Eds.), Geomicrobiology. Science Publishers, Enfield, New Hampshire, USA, pp. 101e137. Amin, A.M., 2007. Fungal Activity for Solubilization and Accumulation of Uranium from Various Grade Ores and Subsequent Chemical Recovery (Ph.D. thesis). Faculty of Science, Menoufia University, Egypt. Amiri, F., Mousavi, S.M., Yaghmaei, S., Barati, M., 2012. Bioleaching kinetics of a spent refinery catalyst using Aspergillus niger at optimal conditions. Biochem. Eng. J. 67, 208e217. Anjum, F., Bahatii, H.N., Ambreen, A., 2009. Bioleaching of black shale by Acidithiobacillus ferrooxidans. Asian J. Chem. 7 (21), 5251e5266. Aung, K.M.M., Ting, Y.P., 2005. Bioleaching of spent fluid catalytic cracking catalyst using Aspergillus niger. J. Biotechnol. 116, 159e170. Brierley, C.L., 2008. How will biomining be applied in future? Trans. Nonferrous Metall. Soc. China 18, 1302e1310. Brierley, C.L., 2010. Biohydrometallurgical prospects. Hydrometallurgy 104, 324e 328. Bromacher, C., Bachofen, R., Brandl, H., 1997. Biohydrometallurgical processing of solids. A patent review. Appl. Microb. Biotechnol. 48, 577e587. Burgstaller, W., Schinner, F., 1993. Mini review: leaching of metals with fungi. J. Biotechnol. 27, 91e116. Chow, N., Nacu, A., Warkentin, D., Alksenov, I., The, H., 2010. The Recovery of Manganese from Low Grade Resource: Bench Scale Metallurgical Test Program
Completed. Kemetco Research Inc., No. 445-5600 Parkwood Way Richmond, BC V6V 2 M2 Canada. Daveis, W., Gray, W.A., 1964. Rapid and specific titrimetric method for the precise determination of uranium using iron II sulphate as reductant. Tolant 11, 1203e 1211. El Aassy, I.E., Ahmed, F.,Y., Afifi, S.Y., Ashami, A.S., 2000. Uranium in Laterites, Southwestern Sinai, Egypt, 1st Seminar on Nuclear Raw Materials and Their Technology, 1e3 Nov, pp. 1e20. El Sayed, H., 2012. Fungal Leaching of Some Heavy Metals from Soil Sediments, South Western Sinai, Egypt (M.Sc. thesis). Faculty of Science, Botany Department, Zagazig University. El Sharkawi, M.A., El Aref, M.M., Abdel Mottelib, A., 1990. Syngenetic and paleokarstic copper mineralization in the paleozoic platform sediments of West Central Sinai, Egypt. Spec. Publ. Int. Ass. Sediment. II, 159e171. Ehrlich, H.L., 2004. Beginnings of rational bioleaching and highlights in the development of biohydrometallurgy: a brief history. Eur. J. Min. Process. Environ. Prot. 4, 102e112. Falk, H., Lavergren, Bergback, B., 2006. Metal mobility in alum shale from Oland Sweden. J. Geochem. Explor. 90, 157e165. Gericke, M., Neale, J.W., Van-Staden, P.J., 2009. A Mintek perspective of the past 25 years in minerals bioleaching. J. SAIMM 109, 567e585. Gilman, J.C., 1957. A Manual Soil Fungi. The Lowa State Univ. Press, Ames, Lowa, USA. Gräfe, M., Power, G., Klauber, C., 2011. Bauxite residue issues III. Alkalinity and associated chemistry. Hydrometallurgy 108 (1e2), 60e79. Harris, N.B., 2005. The deposition of organic-carbon-rich sediments: models, mechanisms, and consequences, introduction. In: Harris, N.B. (Ed.), The Deposition of Organic-carbon-rich Sediments: Models, Mechanisms, and Consequences, Special Publication, 82. Society for Sedimentary Geology, Tulsa, OK, pp. 1e5. Hefnawy, M.A., El-Said, M., Hussein, M., Maisa, A.A., 2002. Fungal leaching of uranium from its geological ores in Alloga area, west central Sinai, Egypt. Online J. Biol. Sci. 2, 346e350. Hefnawy, M.A., Hashad, A.M., Amin, M.M., 2003. Optimization of uranium leaching parameters by A. terreus and P. spinulosum. Afr. J. Mycol. Biotechnol. 1, 17e34. Ibrahim, A.H., Rawia, F.G., Sohair, A.N., Wifkey, A.E., Mamdouh, A.G.H., 2007. Bioleaching of heavy metals by fungi as a technique of ore-dressing applied to orematerials in Sinai, Egypt. In: 10th International Conference of Mining, Petroleum and Metallurgical Engineering, Egypt, pp. 339e410. Johnson, D.B., 2006. Biohydrometallurgy and the environment: intimate and important interplay. Hydrometallurgy 83, 153e166. Kora, M., 1984. The Paleozoic Outcrops of Um Bogma Area, Sinai (PhD thesis). Mansoura University, Mansoura, Egypt, p. 280. Lee, J., Kim, S., Kim, K., Kim, I.S., 2005. Microbial removal of uranium in uranium bearing black shale. Chemosphere 59, 147e154. Liu, Y.G., Zhou, M., Zeng, G.M., Li, X., Xu, W.H., Ting, F., 2007. Effect of solids concentration on removal of heavy metals from mine tailings via bioleaching. J. Hazard. Mater. 141, 202e208. Macaskie, L.E., Dean, A.C.R., 1989. Microbial metabolism, desolubilization and deportion of heavy metals: metal uptake by immobilized cells and application to the detoxification of liquid wastes. Biol. Waste Treat., 159e201. Mohapatra, S., Bohidar, S., Pradhan, N., Kar, R.N., Sukla, L.B., 2007. Microbial extraction of nickel from Sukinda chromite overburden by Acidithiobacillus ferrooxidans and Aspergillus strains. Hydrometallurgy 85, 1e8. Moore, P., 2008. Scaling fresh heights in heap leach technology. Min. Mag. 198, 54e 66. Mulligan, C.N., Kamali, M., Gibbs, B.F., 2004. Bioleaching of heavy metals from a low-grade mining ore using Aspergillus niger. J. Hazard. Mater. 110, 77e84. Murad, A., El-Holi, K., Al-Delaimy, S., 2003. Citric acid production from whely with sugar and additives by Aspergillus niger. Afr. J. Biotechnol. 2 (10), 356e359. Patterson, G.M.L., Boils, C.M., 1995. Regulating of scytophycin accumulation in cultures of Scytonema ocellatum II. Nutrient requirement. Appl. Microbiol. Biotechnol. 43, 692e700. Piper, D.Z., Calvert, S.E., 2009. A marine biogeochemical perspective on black shale deposition. Earth Sci. Rev. 95, 63e96. Pitt, J., 1979. The Genus Penicillium and Teleromorphic State. Eupenicillium and Talaromyces. Academic Press, London, New York, Toronto, Sydney. Ren, W.X., Li, P.J., Yong, G., Li, X.J., 2009. Biological leaching of heavy metals from a contaminated soil by Aspergillus niger. J. Hazard. Mater. 167, 164e169. Santhiya, D., Ting, Y.P., 2005. Bioleaching of spent refinery processing catalyst using A. niger with high yield oxalic acid. J. Biotechnol. 116, 171e184. Shapiro, L., Brannock, W.W., 1962. Rapid analysis of silicate, carbonate and phosphate rocks. U.S. Geol. Surv. Bull. 114, 56e63. Tang, J.A., Valix, M., 2006. Leaching of low grade limonite and nontronite ores by fungi metabolic acids. Miner. Eng. 19, 1274e1279. Umanskii, A.B., Klyushnikov, A.M., 2013. Bioleaching of low grade uranium ore containing pyrite using A. ferrooxidans and A. thiooxidans. J. Radioanal. Nucl. Chem. 295, 151e156. Valix, M., Usai, F., Malik, R., 2001. Fungal bioleaching of low grade laterite ores. Miner. Eng. 14, 197e203. Xu, T.J., Ting, Y.P., 2004. Optimization on bioleaching of incinerator fly ash by Aspergillus niger e use of central composite design. Enzyme Microb. Technol. 35, 444e454. Xu, T.J., Ting, Y.P., 2009. Fungal bioleaching of incineration fly ash: metal extraction and modeling growth kinetics. Enzyme Microb. Technol. 44, 323e328. Yan, G., Viraraghavan, T., 2003. Heavy metal removal from aqueous solution by fungus Mucor rouxii. Water Resour. 37, 4486e4496.
Contents lists available at ScienceDirect
Journal of Environmental Radioactivity journal homepage: www.elsevier.com/locate/jenvrad
Effect of mineral constituents in the bioleaching of uranium from uraniferous sedimentary rock samples, Southwestern Sinai, Egypt Maisa M. Amin a, Ibrahim E. Elaassy a, Mohamed G. El-Feky a, Abdel Sattar M. Sallam b, Mona S. Talaat b, Nilly A. Kawady a, * a b
Nuclear Materials Authority, Cairo, Egypt Faculty of Science Ain Shams University, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 July 2013 Received in revised form 18 February 2014 Accepted 26 February 2014 Available online 28 March 2014
Bioleaching, like Biotechnology uses microorganisms to extract metals from their ore materials, whereas microbial activity has an appreciable effect on the dissolution of toxic metals and radionuclides. Bioleaching of uranium was carried out with isolated fungi from uraniferous sedimentary rocks from Southwestern Sinai, Egypt. Eight fungal species were isolated from different grades of uraniferous samples. The bio-dissolution experiments showed that Aspergillus niger and Aspergillus terreus exhibited the highest leaching efficiencies of uranium from the studied samples. Through monitoring the biodissolution process, the uranium grade and mineralogic constituents of the ore material proved to play an important role in the bioleaching process. The tested samples asserted that the optimum conditions of uranium leaching are: 7 days incubation time, 3% pulp density, 30 C incubation temperature and pH 3. Both fungi produced the organic acids, namely; oxalic, acetic, citric, formic, malonic, galic and ascorbic in the culture filtrate, indicating an important role in the bioleaching processes. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Uranium bioleaching Uraniferous sedimentary rock Aspergillus niger Aspergillus terreus Fungal organic acids
1. Introduction Bioleaching is a specialized biohydrometallurgical process principally based on the ability of microorganisms to transform solid compounds into soluble and extractable elements. The extracted elements could be removed through several techniques (Akcil and Deveci, 2010; Ehrlich, 2004). Metal prices, are not the only motivating factor for employing the biohydrometallurgical process (Brierley, 2008), cost production (Gericke et al., 2009; Moore, 2008) and difficulty in recognition as well as evaluation of ore deposits by conventional processes are among other factors (Brierley, 2010). Fungal leaching of metals from minerals is mainly based upon three synchronous steps. Acidolysis which protonation of oxygen atoms that occurred around the surface of metallic compounds. The proton and oxygen associated with water displace the metal from the surface (Johnson, 2006; Mulligan et al., 2004; Xu and Ting, 2009). Complexolysis, where metal complex formation results in the solubilization of the metal ion e.g. complex of oxalic acid and
* Corresponding author. Tel.: þ20 (0)1008123944. E-mail address: [email protected] (N.A. Kawady). http://dx.doi.org/10.1016/j.jenvrad.2014.02.024 0265-931X/Ó 2014 Elsevier Ltd. All rights reserved.
iron. Furthermore, this process often reduces the toxicity of heavy metals towards fungi (Johnson, 2006). Redoxolysis, when reduction of metal ions occurs in an acidic environment, such as in the case of ferric ions and/or manganese under the influence of oxalic acid (Bromacher et al., 1997). A series of organic acids is formed by fungal metabolism, resulting in acidolysis complex and chelate formation (Mulligan et al., 2004). Lee et al. (2005) described the effect of several factors on uranium bioleaching efficiency of uranium bearing black shale (349 mg kg1 of uranium) using Acidothiobacillus ferrooxidans (iron-oxidizer organism). They observed that the initial inoculations of bacterial cells of batch-type reactors, containing black shale, caused lowering of pH and highering of redox potential as well as of Fe3þ in the aqueous leach solutions over the non-inoculated reactor up to 200 h. Ores are classified into high grade (where metal concentration is relatively high) and low grade (with low concentration of metals, like shale) (Chow et al., 2010). Shales are sedimentary rocks formed during the latter part of the Cambrian period and up to the first part of the Ordovician period, approximately 540e 480 Ma ago (Falk et al., 2006). Their color is commonly tones of gray, brown, green or black due to the presence of some organic pigments in microbes inhibiting the shales. The introduction of quartz causes an increase in the size of their grains and transforms
M.M. Amin et al. / Journal of Environmental Radioactivity 134 (2014) 76e82
them into sandstones. Shale deposits containing the elements Fe, K, Si and Al are most susceptible to microbial transformation (Harris, 2005; Piper and Calvert, 2009). The lower Carboniferous (upper Visean) Um Bogma Formation (Fig. 1) in the southern extreme of Wadi (Vally) Naseib consists of three members, namely lower sandy dolostone, middle siltstone marl intercalations and upper dolostone (Kora, 1984). Normally Karstification process affects the lower (sub-soil) and middle (top soil) to form another soil profile hosting several metallic forms (El Sharkawi et al., 1990). El Aassy et al. (2000) pointed out that lateritization processes affected all three members. These three members are the main target of this study. The aim of this work focused upon using the different biomasses (fungi) isolated from three sedimentary samples (natural habitat) in the bioleaching of uranium from these samples.
77
2. Materials and methods 2.1. Collection of ore samples To represent the Allouga mountain, in southern Wadi Nasieb, three radioactive geologically grapped samples were collected in sterile polyethylene bags from the prospective area in southwestern Sinai, Egypt. These samples (Table 1) were chosen to represent different grades of uranium concentrations. 2.2. Mineralogical and chemical analyses of studied samples An X-ray diffraction technique was used to identify the mineralogical constituent of the study sample using (PHILIPS PW 3710/ 31) diffractometer with automatic sample changer PW 1775, (21
Fig. 1. Geological map of the studied localities, southwestern Sinai, Egypt (After, Alshami, 2003).
78
M.M. Amin et al. / Journal of Environmental Radioactivity 134 (2014) 76e82
at the Regional Center for Mycology and Biotechnology, Al Azhar University, Cairo, Egypt.
Table 1 Samples collected from the three members of Um Bogma Formation. A-1
Sandy dolostone, grey to brown, medium hard, with Cu mineralization (lower member). Grey shale with sulfur patches (upper member). Sandy dolostone with shale interlayer, grey to brown shale (middle member).
A-2 A-3
positions), Scintillation counter, Cu-target tube and Ni filter at 40 kV and 30Am. This instrument is connected to a computer system using X-40 diffraction program and ASTM cards for mineral identification. The pulverized samples were analyzed by a conventional wet chemical technique (Shapiro and Brannock, 1962). The SiO2, Al2O3, TiO2 and P2O5 were constituents recognized using a spectrophotometric method. The contents of Na and K were determined using a flame photometric technique. On the other hand, the total oxides as Fe2O3, MgO and CaO were evaluated by titration methods. The loss on ignition (L.O.I) was calculated gravimetrically. The estimated error for the major constituents is less than 1%. Uranium was analyzed by titration against NH4VO3 (Daveis and Gray, 1964). All the chemical analyses were carried out in the laboratories of the Nuclear Materials Authority, Egypt. 2.3. Microbiological studies The microbial studies include the fungal isolation from the study rock samples and the conditioning of the bioleaching factors as follows: 2.3.1. Media and culture conditions used in bioleaching The media used in this work are Dox agar medium of composition g/L: NaNO3, 2 (0.0325); KH2 PO4, 1 (0.00735); MgSO4$7H2O, 0.5 (0.002033); KCl, 0.5 (0.00676); FeSO4$5H2O trace; sucrose, 30 (0.0877); agar, 15.5 g and yeast extract added to initiate fungal growth. The pH value of the media was adjusted to be 6.5 before autoclaving at 120 C and 1.5 atm. for 20 min. Two techniques were used in fungal isolation from the ore. The first one is the directplating technique, in which fine ore-powder was spread directly on the surface of Dox agar plates under aseptic conditions. The agar plates were incubated at 28 C 2 until the fungal colonies clearly appeared. The second technique is the dilution plating in which 1 g of the ore powder was taken under aseptic conditions and mixed well with 9 ml of sterile distilled water; 0.1 ml of this mixture was spread under aseptic conditions by sterile glass rod on the surface of an agar plate. The plates were incubated at 28 C 2 until development of the colony. The hyphal tips of each colony were removed and plated on the surface of the agar plates. The developed colonies were examined with a microscope to detect contamination. The pure isolated fungi were identified according to Gilman (1957) and Pitt (1979). 2.3.2. Determination of organic acids produced by Aspergillus niger and Aspergillus terreus Both tested organisms were cultivated on Dox liquid media for 7 days at 30 C. Organic acids were recognized in the filtrates by HPLC
2.3.3. Factors affecting the bioleaching of uranium Several factors were investigated in the aim of obtaining the optimum conditions for solubilization of uranium. These factors are fungal activity, pulp densities on fungal growth, incubation periods, incubation temperatures and initial pH values. One hundred ml of Dox liquid medium was placed in 250 ml Erlenmeyer flasks. The flasks supplemented with ore samples concentration. Triplicate sets of flasks were prepared for each organism and ore. The flasks were autoclaved at 1.5 atm for 20 min, after cooling the flasks were inoculated with 1 ml spore suspension. At the end of the incubation period, the culture medium was filtered and centrifuged at 4000 rpm to precipitate more particles, repeated for each factor. The filtrate was kept for uranium determination. 3. Results and discussion 3.1. Mineralogical and chemical analyses of studied samples The mineralogical composition of studied ore samples showed that A-1 consisted mainly of dolomite and gypsum, A-2 consisted of kaolinite and quartz and A-3 consisted of dolomite with ferron dolomite and quartz. The residual mineralogy of the sample A-3 which exhibited high bioleaching efficiency, showed that the rest of the dolomite complexes with the iron (in the original sample) to form ankerite [Ca (MgO.67 FeO.33) (CO3)2]. The chemical analysis of the studied uraniferous sedimentary rocks, which are used in this study, revealed the presence of high contents of SiO2 and Al2O3 in sample A-2 and high contents of CaO, MgO as well as loss on ignition (L.O.I) in samples A-1 and A-3 (Table 2). From this table there are three uranium categories; high grade (A-1) with uranium concentration 800 ppm, moderate grade (A-2) with 660 ppm uranium concentration and (A-3) of the lowest grade uranium concentration 77 ppm. 3.2. Fungal activity on uranium bioleaching An obvious variation in the leaching efficiencies appears with the different investigated fungal species (Table 3) in the following order: Aspergillus species > Penicillium species > Mucor species. Meanwhile, within Aspergillus species the A. niger displayed the highest uranium leachability whereas in Penicillium species, the P. diversum revealed the highest uranium leachability. Variations in bioleaching efficiencies of uranium are strongly influenced by the ore mineralogy, the metal loss through electro-sorption properties of the ore as well as effective and selective leaching by certain strains for some heavy metals more than others (Valix et al., 2001). The optimization parameters affecting bioleaching process were determined upon applying the testes of highly ability fungal strains (A. niger and A. terreus) to uranium solubilization. 3.3. Pulp density and fungal growth inhibition Growth of all Aspergillus species, Penicillium species and Mucor species were noticeably decreased with increasing the pulp density
Table 2 The chemical analyses of the tested samples (wt. %). Sample
SiO2
Al2O3
Fe2O3
MnO
CaO
MgO
Na2O
K2O
P2O5
TiO2
L.O.I
Total
U
A-1 A-2 A-3
17.7 78.6 11.4
4.59 8.67 3.06
0.044 0.032 0.03
0.481 0.02 0.43
22.4 2.24 26.9
15.7 1.6 16.9
0.236 0.18 0.307
0.539 1.173 0.337
0.161 0.615 0.142
0.19 0.53 0.111
37.9 6.32 40.6
99.94 99.99 100.21
0.08 0.066 0.0077
M.M. Amin et al. / Journal of Environmental Radioactivity 134 (2014) 76e82 Table 3 Effect of different ore concentrations (A-1, A-2 and A-3) on the inhibition efficiency (%) of isolated fungi. Fungus sp.
A. niger
A. terreus
A. Fumigatus
A. flavus
P. steckii
P. italicum
P. diversum
Mucor sp.
Sample no.
A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3
Table 4 Bioleaching efficiency of uranium by using the dominant fungal isolates. Fungus species
Sample
Uranium leaching efficiency (%)
Final pH
A. niger
A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3 A-1 A-2 A-3
34 51 57 28 36 42 20 23 33 18 25 28 13 18 21 12 20 25 15 27 33 8 14 22
5.64 5.43 5.38 5.52 5.18 4.74 5.97 6.06 5.63 5.77 6.36 5.43 5.65 5.68 5.11 6.47 6.64 6.81 4.87 5.34 5.65 6.66 6.53 6.21
Inhibition efficiency (%) 1%
2%
4%
6%
8%
16.9 3.1 12.1 10.2 14.6 25.2 1.4 4.3 14.0 4.7 12.1 26.3 0.4 0 15.6 1.3 1.2 5.2 2.7 6.7 16.5 12.0 13.3 12.0
26.3 13.2 32.9 24.7 22.4 28.6 16.3 16.6 15.5 3.3 9.1 28.5 1.5 2.1 20.1 9.9 2.3 11.4 13.9 29.8 21.0 24.0 25.6 33.5
40.4 40.4 53.4 27.7 45.6 44.3 31.4 41.4 44.6 32.5 45.9 59.6 2.6 41.5 30.3 11.2 24.5 27.4 16.4 42.0 45.0 38.9 35.0 44.7
67.6 60.2 65.4 58.5 53.4 57.8 54.8 71.0 58.5 54.0 42.4 61.2 29.2 45.6 37.9 19.6 29.4 38.7 29.9 45.9 51.1 59.5 61.5 69.9
77.1 72.3 77.0 71.9 78.8 78.6 81.6 86.1 67.9 79.9 72.9 71.0 37.4 51.8 41.5 30.2 42.2 41.4 76.2 64.9 68.8 81.3 77.3 73.5
except A. flavus at 2% in both A-1 and A-2 and P. italicum for A-2. After 4% pulp density, the growth of Penicillium species slightly decreased with increasing of the pulp density in the media. This observation suggested that Penicillium species have an ability to grow under stress of uranium concentration more than Aspergillus species. In addition, growth inhibition may be due to increase in the concentration of heavy metals in the leaching environment leading to increase in toxicity, which might have inhibited the growth of the microorganisms (Xu and Ting, 2004). Growth of all isolated fungal strains substantially highly decreased with increasing the concentrations of the used ore samples except P. steckii, P. italicum and P. diversum. This trend changed in the sample A-1 at concentration 4% which led to percentage of growth inhibition that reached 13.8%, 2.5%, 11.2% and 16.4% respectively (Tables 3 and 4). This may be attributed to the ability of this organism to tolerate and grow under stress of uranium. The 1% pulp density apparently proved to be the best for all fungal growth between all concentrations. The highest uranium concentration sample (800 ppm) showed the highest growth in the 1% concentration in A. terrus, A. fumigatus, A. flavus, P. diversum and Mucor sp., while the others A. niger, P. steckii and P. italicum gave high growth in the moderate uranium concentrations (660 ppm). This indicates that the uranium concentrations as well as the lithologic type play an appreciable role in the growth of the fungal species. 3.4. Bioleaching controlling factors The fungal strains of high bioleaching efficiency (A. niger and A. terreus) were selected to determine their bioleaching controlling factors as follows: 3.4.1. Pulp densities Leaching of uranium by fungal strains A. niger and A. terreus increased upon increasing the pulp densities in the growth media up to 3% in the three different grades of uranium ore samples, A-1, A-2 and A-3. The highest percentage of uranium solubilization, on the other hand, resulted from the ore sample with the lowest grade
79
A. terreus
A. fumigatus
A. flavus
P. steckii
P. italicum
P. diversumum
Mucor sp.
of uranium concentration and having ferrous dolomite, as shown in Fig. 2. The best leaching efficiency of uranium using fungal strains occurred at 3% of sample A-3, which reached 80% by A. niger and 56% by A. terreus. At a value 3% pulp density, appears that the leaching efficiency of uranium decreased upon increasing the pulp densities. This may be due to insufficient oxygen transfer, limited nutrition provided to the microbes or deformation of cells in the high solid concentrations, as described by Mohapatra et al. (2007). The decrease in the bioleaching efficiency by increasing the sample concentrations may be due to the final pH value which was found to increase with increasing the sample concentrations (but still less than the initial pH of the medium). This led to an increase in the binding capacity of the fungal mycelium at the higher concentrations, as mentioned by Hefnawy et al. (2002). Meanwhile, this led to the decrease of metal concentrations in the actual leaching liquor and the decrease of the solubilization efficiency, as investigated by Tang and Valix (2006). Santhiya and Ting (2005), Aung and Ting (2005) and Liu et al. (2007) mentioned that the optimum pulp density in the bioleaching was 1% (w/v). On the other hand, Ibrahim et al. (2007) obtained the maximum solubilization of uranium and alumina at 4% (w/v) from gibbsite, dolostone and mudstone ores by A. niger. 3.4.2. Incubation periods Uranium leaching by A. niger was highly affected by the incubation periods. The best solubilization activity occurred upon 7 days of incubation; where the amounts of uranium, solubilized from the samples A-1, A-2 and A-3, were 45.7%, 56% and 64% respectively. However, after 7 days of incubation the amounts of uranium solubilized slightly decreased with increasing incubation periods up to 10 days. The same pattern of solubilization of uranium by A. niger was followed by A. terreus and resulted in 27%, 35% and 47% leaching efficiency for A-1, A-2 and A-3, respectively (Fig. 3). The amount of uranium solubilized in the growth media slightly decreased with increasing the incubation period above 7 days. These results were in agreement with those obtained by El Sayed (2012), where eight and six days were the optimum incubation periods for A. niger and A. fumigatus to solubilize uranium and REEs from gibbsite-bearing shale sample.
80
M.M. Amin et al. / Journal of Environmental Radioactivity 134 (2014) 76e82
Fig. 2. Relation between different Pulp densities and Uranium Solubilization by A. niger and A. terreus at 30 C for 7 days: (a) A-1, (b) A-2 and (c) A-3.
Fig. 3. Relation between different Incubation Periods and Uranium Solubilization by A. niger and A. terreus at 30 C: (a): A-1, (b): A-2 and (c): A-3.
3.4.3. Incubation temperatures Uranium leaching from its ores was found to be highly affected by the incubation temperature; it reached its maximum at 30 C. At this temperature, A. niger solubilized 43%, 54% and 64% of uranium found in the ore samples A-1, A-2 and A-3 respectively (Fig. 4a, b and c). Whereas, uranium leached by A. terreus at 30 C revealed 26%, 34.8% and 45% solubilization respectively. At this incubation temperature, the final pH value of the medium shifted toward acidity. El Sayed (2012) mentioned that the maximum bioleaching of heavy metals occurred at 30 C for A. niger and 35 C for A. fumigatus. At higher temperatures (40 C and 45 C), though, the metabolic rate of organism decreases and the organism might even not survive (Mohapatra et al., 2007).
pH 3 is apparently the best for bioleaching efficiency of uranium. It reached 65% and 49.3% for the 1% sample A-3 by A. niger and A. terreus respectively. Moreover, the increasing of the growth media pH value led to an increase in the binding capacity of the fungal cells as well as a decreasing the leaching efficiency of uranium (Hefnawy et al., 2003), yet still less than the initial pH value of the medium. Amin (2007) suggested that the maximum solubilization of uranium was obtained at the initial pH value of 4. The variation in the optimum initial pH value of metal solubilization may be due to the different chemistry of the metal complexation, as previously described by Macaskie and Dean (1989); Yan and Viraraghavan (2003). The pH value of the medium is very important for the growth of microorganisms, for the character of their metabolism and hence for the biosynthesis of their antimicrobial products due to secondary metabolites (Patterson and Boils, 1995).
3.4.4. Initial pH The optimum pH value was determined by using the fungal strains which have the highest leachability at 1% from samples A-1, A-2, and A-3 at 30 C for 7 days on Dox-liquid media. Fig. 5 showed that the amount of released uranium decreased with increasing the initial pH value of the growth media. The highest bioleaching efficiency of uranium from the samples was obtained at pH 3 by A. niger followed by A. terreus. The final pHs increased with increasing initial pH values of the growth medium. The optimum
3.5. Role of organic acids and mineral constituents The application of bioleaching technology to uranium leaching results in an additional acid generation as a consequence of microbial activity. Thus leads to a decrease of acid consumption at uranium industrial plants (Umanskii and Klyushnikov, 2013).
M.M. Amin et al. / Journal of Environmental Radioactivity 134 (2014) 76e82
81
Fig. 5. Relation between different Initial pHs and Uranium Solubilization by A. niger and A. terreus for 7 days: (a) A-1, (b) A-2 and (c) A-3. Fig. 4. Relation between different Incubation Temperatures and Uranium Solubilization by A. niger and A. terreus for 7 days: (a): A-1, (b): A-2 and (c): A-3.
Acidolysis is the principal mechanism in bioleaching of metals by microbes which produces organic acids such as citric, oxalic, gluconic and malic during bioleaching processes (Johnson, 2006). Organic acids dissolve metals from minerals by displacement of metal chelates (Ren et al., 2009). Accordingly, different mineral constituents of tested ore samples with different binding chelation need different types and concentrations of produced organic acids. This, on the other hand, played a direct and important role in the bioleaching process, and confirms that depletion of bioleaching agents could be one reason in decreasing the rate of metal extraction (Amiri et al., 2012). There are two important functions in bioleaching. First, they can facilitate the dissolution of metal ions from leaching materials through chelation of the metals released in solution and also destabilize the bonds between the surface metal and bulk leaching materials (Gräfe et al., 2011). Second, they can reduce the adverse effects of metal ions imposed on microorganisms through chelation or complexation (Burgstaller and Schinner, 1993). Metabolites of both fungal strains were subjected to proper chromatographic analysis (Fig. 6a and b). The analysis proved that A. niger secretes, in the growth media, organic acids such as, formic, acetic, citric and oxalic. This agrees to great extent with previous,
A. niger generating organic acids like oxalic, citric, malic and tartaric, which are effective for metal solubilization (Anjum et al., 2009; Mulligan et al., 2004; Murad et al., 2003). Whereas, A. terreus secreted citric, oxalic, galic, malonic and ascorbic as secondary metabolites, which were effective in uranium solubility. The organic acids produced by A. niger and A. terreus under 1% and 5% bioleaching processes are shown in Fig. 6. These acids are essential for the leaching of heavy metals from ore materials and solid wastes (Ren et al., 2009; Santhiya and Ting, 2005). Oxalic acid is the major lixiviate among the metabolites product by A. niger, which yielded highest extraction of metals at 1% pulp density in 60 days (Santhiya and Ting, 2005). These dissoluted metals were related to different mineral constituents of the ore samples. 4. Conclusions The following conclusions were drawn from this study: 1 The eight fungal species were identified as A. niger, A. terreus, A. fumigatus, A. flavus, P. italicum, P. diversum, P. steckii and Mucor species. A. niger and A. terreus were the only isolates which achieved the highest leaching efficiency of uranium from the studied uraniferous samples.
82
M.M. Amin et al. / Journal of Environmental Radioactivity 134 (2014) 76e82
Fig. 6. Typical organic acids chromatograph in fungi grown on Dox liquid medium (a) A. niger (1: formic acid; 2: oxalic acid; 3: citric acid and 4: acetic acid) and. (b) A. terreus, (1: oxalic acid; 2: citric acid; 3: ascorbic acid; 4: malonic acid; and 5: galic acid).
2 All tested fungi showed variations in bioleaching directly related to variations in mineral constituents and uranium grades. 3 The optimum conditions of uranium leaching from its ores, using A. niger and A. terreus, were found to be 7 days incubation time, 3% pulp density pH 3 and 30 C incubation temperature. 4 A. niger and A. terreus exhibit an appreciable potential in generating a variety of organic acids which play an important and effective role for uranium leaching. References Akcil, A., Deveci, H., 2010. Mineral biotechnology of sulphides (Chapter 4). In: Jains, S., Khan, A., Rai, M.K. (Eds.), Geomicrobiology. Science Publishers, Enfield, New Hampshire, USA, pp. 101e137. Amin, A.M., 2007. Fungal Activity for Solubilization and Accumulation of Uranium from Various Grade Ores and Subsequent Chemical Recovery (Ph.D. thesis). Faculty of Science, Menoufia University, Egypt. Amiri, F., Mousavi, S.M., Yaghmaei, S., Barati, M., 2012. Bioleaching kinetics of a spent refinery catalyst using Aspergillus niger at optimal conditions. Biochem. Eng. J. 67, 208e217. Anjum, F., Bahatii, H.N., Ambreen, A., 2009. Bioleaching of black shale by Acidithiobacillus ferrooxidans. Asian J. Chem. 7 (21), 5251e5266. Aung, K.M.M., Ting, Y.P., 2005. Bioleaching of spent fluid catalytic cracking catalyst using Aspergillus niger. J. Biotechnol. 116, 159e170. Brierley, C.L., 2008. How will biomining be applied in future? Trans. Nonferrous Metall. Soc. China 18, 1302e1310. Brierley, C.L., 2010. Biohydrometallurgical prospects. Hydrometallurgy 104, 324e 328. Bromacher, C., Bachofen, R., Brandl, H., 1997. Biohydrometallurgical processing of solids. A patent review. Appl. Microb. Biotechnol. 48, 577e587. Burgstaller, W., Schinner, F., 1993. Mini review: leaching of metals with fungi. J. Biotechnol. 27, 91e116. Chow, N., Nacu, A., Warkentin, D., Alksenov, I., The, H., 2010. The Recovery of Manganese from Low Grade Resource: Bench Scale Metallurgical Test Program
Completed. Kemetco Research Inc., No. 445-5600 Parkwood Way Richmond, BC V6V 2 M2 Canada. Daveis, W., Gray, W.A., 1964. Rapid and specific titrimetric method for the precise determination of uranium using iron II sulphate as reductant. Tolant 11, 1203e 1211. El Aassy, I.E., Ahmed, F.,Y., Afifi, S.Y., Ashami, A.S., 2000. Uranium in Laterites, Southwestern Sinai, Egypt, 1st Seminar on Nuclear Raw Materials and Their Technology, 1e3 Nov, pp. 1e20. El Sayed, H., 2012. Fungal Leaching of Some Heavy Metals from Soil Sediments, South Western Sinai, Egypt (M.Sc. thesis). Faculty of Science, Botany Department, Zagazig University. El Sharkawi, M.A., El Aref, M.M., Abdel Mottelib, A., 1990. Syngenetic and paleokarstic copper mineralization in the paleozoic platform sediments of West Central Sinai, Egypt. Spec. Publ. Int. Ass. Sediment. II, 159e171. Ehrlich, H.L., 2004. Beginnings of rational bioleaching and highlights in the development of biohydrometallurgy: a brief history. Eur. J. Min. Process. Environ. Prot. 4, 102e112. Falk, H., Lavergren, Bergback, B., 2006. Metal mobility in alum shale from Oland Sweden. J. Geochem. Explor. 90, 157e165. Gericke, M., Neale, J.W., Van-Staden, P.J., 2009. A Mintek perspective of the past 25 years in minerals bioleaching. J. SAIMM 109, 567e585. Gilman, J.C., 1957. A Manual Soil Fungi. The Lowa State Univ. Press, Ames, Lowa, USA. Gräfe, M., Power, G., Klauber, C., 2011. Bauxite residue issues III. Alkalinity and associated chemistry. Hydrometallurgy 108 (1e2), 60e79. Harris, N.B., 2005. The deposition of organic-carbon-rich sediments: models, mechanisms, and consequences, introduction. In: Harris, N.B. (Ed.), The Deposition of Organic-carbon-rich Sediments: Models, Mechanisms, and Consequences, Special Publication, 82. Society for Sedimentary Geology, Tulsa, OK, pp. 1e5. Hefnawy, M.A., El-Said, M., Hussein, M., Maisa, A.A., 2002. Fungal leaching of uranium from its geological ores in Alloga area, west central Sinai, Egypt. Online J. Biol. Sci. 2, 346e350. Hefnawy, M.A., Hashad, A.M., Amin, M.M., 2003. Optimization of uranium leaching parameters by A. terreus and P. spinulosum. Afr. J. Mycol. Biotechnol. 1, 17e34. Ibrahim, A.H., Rawia, F.G., Sohair, A.N., Wifkey, A.E., Mamdouh, A.G.H., 2007. Bioleaching of heavy metals by fungi as a technique of ore-dressing applied to orematerials in Sinai, Egypt. In: 10th International Conference of Mining, Petroleum and Metallurgical Engineering, Egypt, pp. 339e410. Johnson, D.B., 2006. Biohydrometallurgy and the environment: intimate and important interplay. Hydrometallurgy 83, 153e166. Kora, M., 1984. The Paleozoic Outcrops of Um Bogma Area, Sinai (PhD thesis). Mansoura University, Mansoura, Egypt, p. 280. Lee, J., Kim, S., Kim, K., Kim, I.S., 2005. Microbial removal of uranium in uranium bearing black shale. Chemosphere 59, 147e154. Liu, Y.G., Zhou, M., Zeng, G.M., Li, X., Xu, W.H., Ting, F., 2007. Effect of solids concentration on removal of heavy metals from mine tailings via bioleaching. J. Hazard. Mater. 141, 202e208. Macaskie, L.E., Dean, A.C.R., 1989. Microbial metabolism, desolubilization and deportion of heavy metals: metal uptake by immobilized cells and application to the detoxification of liquid wastes. Biol. Waste Treat., 159e201. Mohapatra, S., Bohidar, S., Pradhan, N., Kar, R.N., Sukla, L.B., 2007. Microbial extraction of nickel from Sukinda chromite overburden by Acidithiobacillus ferrooxidans and Aspergillus strains. Hydrometallurgy 85, 1e8. Moore, P., 2008. Scaling fresh heights in heap leach technology. Min. Mag. 198, 54e 66. Mulligan, C.N., Kamali, M., Gibbs, B.F., 2004. Bioleaching of heavy metals from a low-grade mining ore using Aspergillus niger. J. Hazard. Mater. 110, 77e84. Murad, A., El-Holi, K., Al-Delaimy, S., 2003. Citric acid production from whely with sugar and additives by Aspergillus niger. Afr. J. Biotechnol. 2 (10), 356e359. Patterson, G.M.L., Boils, C.M., 1995. Regulating of scytophycin accumulation in cultures of Scytonema ocellatum II. Nutrient requirement. Appl. Microbiol. Biotechnol. 43, 692e700. Piper, D.Z., Calvert, S.E., 2009. A marine biogeochemical perspective on black shale deposition. Earth Sci. Rev. 95, 63e96. Pitt, J., 1979. The Genus Penicillium and Teleromorphic State. Eupenicillium and Talaromyces. Academic Press, London, New York, Toronto, Sydney. Ren, W.X., Li, P.J., Yong, G., Li, X.J., 2009. Biological leaching of heavy metals from a contaminated soil by Aspergillus niger. J. Hazard. Mater. 167, 164e169. Santhiya, D., Ting, Y.P., 2005. Bioleaching of spent refinery processing catalyst using A. niger with high yield oxalic acid. J. Biotechnol. 116, 171e184. Shapiro, L., Brannock, W.W., 1962. Rapid analysis of silicate, carbonate and phosphate rocks. U.S. Geol. Surv. Bull. 114, 56e63. Tang, J.A., Valix, M., 2006. Leaching of low grade limonite and nontronite ores by fungi metabolic acids. Miner. Eng. 19, 1274e1279. Umanskii, A.B., Klyushnikov, A.M., 2013. Bioleaching of low grade uranium ore containing pyrite using A. ferrooxidans and A. thiooxidans. J. Radioanal. Nucl. Chem. 295, 151e156. Valix, M., Usai, F., Malik, R., 2001. Fungal bioleaching of low grade laterite ores. Miner. Eng. 14, 197e203. Xu, T.J., Ting, Y.P., 2004. Optimization on bioleaching of incinerator fly ash by Aspergillus niger e use of central composite design. Enzyme Microb. Technol. 35, 444e454. Xu, T.J., Ting, Y.P., 2009. Fungal bioleaching of incineration fly ash: metal extraction and modeling growth kinetics. Enzyme Microb. Technol. 44, 323e328. Yan, G., Viraraghavan, T., 2003. Heavy metal removal from aqueous solution by fungus Mucor rouxii. Water Resour. 37, 4486e4496.