FUNGAL LEACHING OF MANGANESE ORE

Transcription

1 Trans. Indian Inst. Met. Vol.57, No. 5, October 2004, pp TP 1908 FUNGAL LEACHING OF MANGANESE ORE C. Acharya, R.N. Kar, L.B. Sukla and Vibhuti N. Misra Regional Research Laboratory, Bhubaneswar celinacharya@hotmail.com (Received 16 April 2004 ; in revised form 30 June 2004) ABSTRACT Bioleaching is technologically feasible for extraction of manganese from low grade ores (containing manganese less than 35% by weight). The principle involves the non-enzymatic reduction of pyrolusite [Mn(IV) oxides] to +2 oxidation state by fungi with the production of metabolites such as oxalic acid and citric acid. In the present investigation, a fungal strain, Penicillium citrinum was isolated from top soil of Joda East manganese mine area, Tata Iron and Steel Company(TISCO), Orissa. Growth of the fungus was determined in terms of the final ph of the growth medium, biomass dry weight and total acid produced by the fungus. These data were used to evaluate the growth kinetics. Finally, it was used for bioleaching of low grade manganese ore. Effect of various parameters on in-situ leaching of manganese ore with P. citrinum such as (a) particle size (b) pulp density (c) sucrose concentration (d) inoculum size and (e) duration of leaching were studied. The maximum solubilisation of manganese (64.6%) was obtained with particle size of -45µm of the ore at pulp density of 2% (w/v), sucrose concentration 10% (w/v) and inoculum size of 10% (v/v) in a period of 30 days. 1. INTRODUCTION Since no quality steel can be produced without the addition of small amount of manganese, it is aptly called the Achilles heel of the iron and steel industry. Manganese constitutes 0.10% by weight of earth s crust 1. About 95% of total production of Mn ore is used in iron and steel production. The major accumulating minerals of manganese at present, are in the form of oxides, carbonates and silicates; the most important being psilomelane, pyrolusite, manganite, rhodochrosite, chondrite, etc. Usually, manganese ore is classified into three grades according to the manganese content of the ore high grade contains 44-48% and above Mn, medium grade 35-44% and low grade 25-35% Mn respectively 2. Recovery of metals from low-grade ores using existing pyrometallurgical and hydrometallurgical technologies are prohibitively expensive due to high energy and capital costs. Another major problem in many parts of the world is the rising cost of environmental protection. The recovery of manganese from manganiferous ores using bioleaching with different microorganisms is considered a better alternative as it holds the promise of lowering the fixed capital costs and of reducing environmental pollution. The microbial leaching of manganese in general is carried out by heterotrophic microorganism which live in microaerobic conditions 3 and require one or more organic nutrients to serve as energy and carbon sources for their growth. 2. EXPERIMENTAL 2.1 Manganese Ore Low grade manganese ore, used for the studies, was collected from Joda East manganese mines of Tata Iron & Steel Company Ltd. in Keonjhar district in Orissa. The ore assayed 25.7% of manganese and 25% of iron. 2.2 Microorganisms Isolation of microorganisms: Nearly eight strains were isolated from the top soil near Joda East manganese mine area. All of the eight strains were purified by repeated subculturing and single colony isolation on agar media. Later,

2 TRANS. INDIAN INST. MET., VOL. 57, NO. 5, OCTOBER 2004 they were transferred to mineral salt medium for studies of their growth and leaching characteristics Screening of microorganism for leaching efficiency Submerged cultivation technique 4 was carried out for screening microorganisms according to their manganese solubilizing efficiency. 90 ml of mineral salt medium containing (g/l): NH 4 NO 3, 3.0; KH 2 PO 4, 1.0; MgSO 4. 7H 2 O, 0.5; and sucrose, 100, ph adjusted to 6.5 with dilute NaOH, was taken in 250 ml Erlenmeyer flasks. Growth of the microorganisms as well as the bioleaching studies were done using the mineral salt media. 10 ml of inoculum of various strains was added separately to the flasks containing sterilised media and 2% (w/v) of manganese ore (- 500 µm). The flasks were incubated at 32 o C with shaking (140 rpm) for 10 days. After 10 days, the flasks were sterilized and the contents were filtered using Whatman filter paper. The solubilized manganese in the filtrate in each case was quantified by Perkin Elmer (3100) atomic absorption spectrophotometer. The microorganism which showed maximum solubilisation of manganese, was chosen for further bioleaching studies. Further, selection of the microorganism capable of solubilizing manganese was also observed using direct screening method 4. In this method, all the eight colonies, previously isolated, were purified by single colony streaking. Each organism was restreaked on solid media impregnated with sterile, sub-micron, ball-milled manganese ore. The species, which removed suspended ore particles and produced halos around colonies growing on agar plates, was sub-cultured for taxonomic identification Identification: Nearly eight strains (five fungal strains and three bacterial strains) were isolated from the top soil of manganese mine area. Studies on screening of the microbes with maximum efficiency for manganese solubilization indicated that a fungus, identified as Penicillium citrium Thom, was best suited for this purpose. It was identified by Institute of Microbial Technology, Chandigarh (MTCC 3303). Hence, the growth and bioleaching studies were performed using that fungus Growth studies: For growth studies of Penicillium citrinum, two sets of experiments were carried out, one set in shaking condition (140 rpm) and the other in static condition. 90ml of sterilized mineral salt media was taken with 10ml of inoculum (10 6 spores/ml) in 250ml Erlenmeyer flasks. The flasks were incubated at 32 o C. Growth, in both the cases, was measured at an interval of 3 days. At an interval of 3 days, the flasks were sterilized and the ph was noted with a glass electrode. The contents were filtered using a pre-weighed Whatman No.1 filter paper. The biomass which remained on the pre-weighed filter paper was dried at 100 o C for 1 hr. Then the dry weight of the biomass was measured in a single pan balance 5. The filtrate was used for determination of total acid strength 6. Thus, the growth of Penicillium citrinum was determined in terms of final ph, biomass dry weight and total acid produced. 2.3 Bioleaching Studies Leaching experiments were done in 250 ml Erlenmeyer flask containing 90 ml of sterilized mineral salt medium, 10 ml of fungal inoculum with known quantity of manganese ore. Flasks were incubated at 32 O C with shaking (140 rpm). Effect of various parameters were studied. Size fractions of manganese ore varied from µm to -45µm. The pulp density, inoculum size and sucrose concentration varied from 2-10%. Experiments were carried out for a period of 30 days. To study the effect of time period on bioleaching of manganese ore, a sample of size µm was leached at 2% (w/v) pulp density, 10% (v/v) inoculum and 10% (w/v) of sucrose. Experiments were run for 90 days. At the end of each experiment, the final ph was measured after sterilization of the leaching vessel. The contents of the same were filtered. The biomass and the ore which remained on the filter paper were thoroughly washed with distilled water and then with dilute sulphuric acid. The washings were added to the filtrate. Thus the overall manganese extraction in each case, was taken to be the sum of acid desorbed and soluble manganese at the termination of each experiment. Analyses of overall manganese extracted using P. citrinum were carried out by Atomic Absorption Spectrophotometer using suitable dilutions. 502

3 ACHARYA, et.al., : FUNGAL LEACHING OF MANGANESE ORE 3. RESULTS AND DISCUSSION 3.1 Growth Studies Growth of P. citrinum in shaking condition: In shaking condition (140 rpm), it was observed that the ph of the media fell from 6.5 to 3.7 during growth of the fungus at 32 o C. The metabolic products that are end products of their respiratory or energy metabolism are mostly organic acids. These acids, when produced during the growth of the fungal culture tend to reduce the ph of the media 4. The ph of the growth media is inversely related to the strength of the acid produced. It can be observed from Fig.1 that as the ph is lowered during exponential phase of the fungus (3-12 days), the strength of acid production increased to 0.08N. After 12 days, the ph of the fungal culture exhibited a modest rise and correspondingly, the strength of the acid in the growth media also marginally decreased. Finally, after 18 days, there is an onset of death phase of the culture. The assumption is that the amount of acid produced under specified conditions and during a fixed period of time, is proportional to the magnitude of fungal population or biomass, though sometimes, the dry weight may continue to increase without corresponding cell growth. During the exponential growth phase, the biomass continues to increase from 0.09g/l to 9.04 g/l in 12 days (Fig. 2). Fig. 1 : Variation of ph and acidity with time in P. citrinum in shaking condition. Fig. 2 : Biomass dry wt. (g/l) of P. citrinum vs. time in static and shaking conditions. 503

4 TRANS. INDIAN INST. MET., VOL. 57, NO. 5, OCTOBER 2004 First order rate constants (k) were derived for growth of the fungus (in terms of biomass). The reaction of growth followed the integrated simple first order rate expression: -1n (y) = kt (1) where y,t and k are total biomass, time and rate constant respectively. The rate constant was evaluated for growth of shaking culture of P. citrinum by fitting Eq.1 to the data in Fig.2. The growth rate (in terms of dry wt. of biomass) displayed linearity in the experimental growth phase of the fungus. The slope was calculated, using linear regression programme for determination of growth rate constant (k). It was observed to be 0.13 day -1 with a correlation coefficient of 0.91 which indicated a good linear fit Growth of P. citrinum in static condition: Growth of the fungus in static condition was observed to be higher than in shaking condition in terms of ph, strength of total acid produced and biomass dry weight. The ph falls from 6.5 to 3.5 in just nine days. Correspondingly, the strength of total acid produced by the fungal culture was found to be 0.1N in the same period from 0.02N (Fig.3). The exponential phase of the fungus was up to 9 days after which there is slight rise in ph and reduction in the strength of the total acid generated in the growth media. When cells grow in stationary liquid medium, the nutrients around them are rapidly depleted. In our study, a fair amount of growth, indicated by a yield of sizeable amount of dry mycelial weight (biomass) was observed within 9 days period. As high as 11.2 g/l of biomass was obtained in this period (Fig.2). The strength of the total acid produced by the static fungal culture during this period was 0.1N which supported the assumption of Pelczar et al. 7 that amount of acid produced by any fungus under specified conditions was proportional to the magnitude of the fungal biomass. Following the end of growth phase, marked by cessation of uptake of nitrogen, a maintenance or a stationary phase occurs when the culture metabolizes sugar, or possibly fat. Low oxygen concentrations affect the metabolism of the cells and production of alcohol, butanediol and volatile metabolites replaces the growth and carbon dioxide production 8. As a result, there is increase in ph of the growth media during stationary phase as can be observed from Fig.3. This is followed by the death phase where the mycelium appears to become exhausted due to steady decay of enzymes and proteins. The breakdown of cells which occurs at this time, is associated with foaming which was observed in our studies at the end of the experiment. First order rate constant was computed for growth of static culture of Penicillium citrinum by fitting Eq.1 to the data in Fig.2. The growth rate constant (k) was found by calculating the slope of the plot of 1n(y) (where y is biomass dry weight) against time. Fig. 3 : Variation of ph and acidity with time in P. citrinum in static condition. 504

5 ACHARYA, et.al., : FUNGAL LEACHING OF MANGANESE ORE It was observed to be 0.18 day -1 with a correlation coefficient of 0.9 which confirmed a good fit. Though it was observed from the present study that the growth and therefore, biomass yield was better in case of stationary submerged cultures of P. citrinum, shaken or agitated submerged cultures were chosen for bioleaching of manganese ore because P. citrinum is an aerobic organism. For aerobic cultures, oxygen must be supplied at least by shaking through the liquid. In absence of oxygen or in low oxygen concentration in the growth media, the organism changes its metabolism to a system more suited to low oxygen conditions. Also, when cells grow in stationary liquid medium, the nutrients of the media are rapidly depleted producing large amount of different acids. The ageing of such culture is also rapid Bioleaching studies As described earlier, the bioleaching experiments were carried out in 250ml Erlenmeyer flasks containing mineral salt media, inocula (10 6 spores/ ml) and manganese ore. The flasks were incubated at 32 o C on reciprocal shaker at 140 rpm. The influence of various parameters like particle size, pulp density, sucrose concentration, inoculum size and duration on bioleaching of manganese ore were studied. The microbial leaching of manganese ore with heterotrophic microorganisms like fungi generally follows an indirect mechanism 10,11 where manganese solubilization is due to reduction. In all cases, the biological process occurs in presence of sacchariferous substances. The overall reactions involved in this process: 24 MnO 2 + C 12 H 22 O H + = 24 Mn CO H 2 O (1) Mn 2+ + CO 2 + H 2 O = MnCO 3 + 2H + (2) It was observed that the total manganese extraction (soluble and acid desorbed manganese) is maximum (64.6%) for finest particle size of -45µm. It is due to the accessibility of the reaction zones within ore particle to the fungus. It was observed that soluble manganese in the culture fluids was in the range of 4.1% to 19.8% for different granulometric size range. XRD data of the manganese ore in our investigation indicated the presence of more pyrolusite than other minerals like cryptomelane, rhodochrosite, and calcite. Manganese oxides containing relatively more pyrolusite were less susceptible to microbial attack. 11 A series of experiments were carried out with different pulp density suspensions varying from 2 to 10% (w/v) manganese ore in inoculated runs. The particle size of -500µm was maintained for the experiments. The highest dissolved or solubilized manganese concentration (13.8%) was achieved with 2% (w/v) pulp density at 32 o C. Higher Mn 2+ extraction yields are achieved with lower pulp densities. Similar observations were made by Veligo et al., The total recovery of 33% was maximum for 2% (w/v) of manganese ore as well as total iron recovered was 6.8%. With the increase in pulp density, the manganese extraction yields, decreased. It may be due to unavailability of oxygen or nutrients to the fungus. The experiments conducted to observe the influence of sucrose concentration on bioleaching of manganese ore indicated that there was a direct relation between manganese extraction and the total amount of sucrose present in the media. Heterotrophs like Penicillium sp. were found to solubilize MnO 2 by producing citric and oxalic acids in the culture medium 13. The culture medium used in these tests contained sucrose. This type of media is commonly utilized in microaerobial monitored conditions. In our investigation, it was observed that a maximum of 33% of total recovery of manganese was obtained with 10% sucrose with the sample size of -500µm. The manganese extraction yields were increased as the sucrose concentration in the culture media increased. In the control (media without sucrose) there was a low recovery of Mn 2+ (6.98%) and iron (0.9%). In the absence of energy substrate like sucrose Penicillium citrinum did not produce organic acids which could reduce MnO 2 and hence, a low recovery was observed. It is observed from our studies that the product formation (manganese reduction) is related mainly to the microbial inoculum or growth (product associated to the growth). The more the inoculum, more is the production of organic acids which eventually leads to manganese reduction. An optimum of 10% (v/v) inoculum of fungal spores gave the 505

6 TRANS. INDIAN INST. MET., VOL. 57, NO. 5, OCTOBER 2004 maximum total extraction of 33% of manganese with -500µm sample size (Fig.4). One of the major drawbacks with submerged cultivation technique with fungus is that the leaching material is adsorbed to fungal mycelium 4. More than 10% (v/v) inoculum was not taken for our investigation as it would have caused unnecessary clumping of the mycelia and the adsorption of leached manganese to the mycelia. The effect of time on bioleaching of manganese ore with P. citrinum was studied for a period of 90 days using finer particle size of µm. The studies were conducted with an initial ph of 6.5 at 32 o C. As the experiments proceeded, manganese was leached out or extracted from the ore by indirect mechanism or non-enzymatically by the fungus P. citrinum. The ph of the leach liquor fell to 5.25 in 30 days giving a total extraction of 58% [16.1% of soluble manganese(fig.5) and 41.9% of acid washed manganese in the leach system]. There was a net acid consumption at the time of maximum Mn 2+ reduction. A recovery of 10.3% iron was observed within a period of 30 days. There was an increasing trend observed in total manganese recovery (68.3% out of which 25.1% is soluble manganese) till 45 days. After 45 days, there was a decrease in the total recovery of manganese (59.5% out of which 24.1% is soluble manganese) as well as iron (6%). May be the manganese which is leached out gets accumulated on the surface of some structure of the fungal hyphae 14. It was observed during the bioleaching of manganese ore that the leaching was growth independent and most probably the result of microaerobic fermentation where the reductive process was associated with the production of reductive compounds 15. The reduction in the recovery of manganese after 45 days may be due to: Fig. 4 : Influence of inoculum size on total manganese extraction. Fig. 5 : % Mn extraction during in-situ leaching(soluble manganese) 506

7 ACHARYA, et.al., : FUNGAL LEACHING OF MANGANESE ORE (1) the accumulation of leached material (manganese) on the surface of fungal hyphae 14. (2) the production of volatile metabolites instead of organic acids and decay of the enzymes of the fungus following its death phase 8 as a result of which there is a slight rise in ph of the media. The experiments were terminated at the end of 90 days following a reduction in the manganese extraction yield. 4. CONCLUSIONS Manganese solubilizing microorganisms can be isolated from a variety of microbial habitat including active or abandoned mine sites. In our studies, Penicillium citrinum was isolated from the top soil of an active mine site of Joda East manganese mines of TISCO. The growth studies of the identified fungus were performed in static as well as in shaking conditions. The growth was measured in terms of final ph, strength of total acid produced by the fungus and the biomass dry weight of the fungus. Growth of P. citrinum was better in static condition where the strength of total acid produced during the exponential phase was 0.1N and the biomass dry weight was 11.2 g/l. Whereas in shaking condition, the maximum strength of the total acid produced was 0.08N in exponential phase and the biomass dry weight during that period is 9.04 g/l. First order rate constants (k) were derived for growth of the fungus (in terms of biomass). The rate constant for the fungus shaking condition was found to be 0.13 day -1 and in static condition it was higher, i.e., 0.18 day -1. Effect of various parameters on in-situ leaching of manganese ore with P. citrinum such as particle size, pulp density, sucrose concentration, inoculum size, were studied for a duration of 30 days. The conditions optimized for maximum recovery of manganese (64.6%) were observed to be: particle size, -45 µm; pulp density 2% (w/v); sucrose concentration, 10% (w/v); inoculum size 10% (v/v), duration : 30 days. ACKNOWLEDGEMENT The first author wishes to thank CSIR for granting her a Research Associateship. REFERENCES 1. Alexandrov E A, Manganese : element and geochemistry. In : (ed) Fairbridge R W, Encyclopedia of Geochemistry and Environmental Sciences, Vol.IVA, Van Nestrand, Reinhold, New York (1972) Reddy A J, An overview of Manganese ore and Alloys Industry in India in a Global Perspective, Seminar Proceedings, Manganese Ores and Alloys, organized by Society of Geoscientists and Allied Technologies (1995). 3. Ehrlich H L, Manganese as an energy source for bacteria. In: (ed) Nrigau, J O, Environmental Biogeochemistry, An Arbor Press (1976) Burgastaller W and Schinner F, Leaching of metals with fungi, J Biotechnol 27 (1993) Swamy K M, Sukla L B, Narayana K L, Kar R N and Panchanadikar V V, Application of ultrasonics in improvement of fungal strains, Acoustic Lett 17 (1993) Vogel A I, A text book of quantitative inorganic analysis including Elementary and Instrumental Analysis, IIIrd ed, The English language Book Society and Longmans Green & Co Ltd (1962). 7. Pelczar M J Jr, Chan E C S, Krieg N R and Pelczar M F, Microbiology, Tata McGraw Hill Publishing Company Limited, New Delhi (1993). 8. Harrison D E F and Pirt S J, Automatic control of dissolved oxygen concentration in stirred microbial cultures, J Gen. Microbiol 46 (1967) Kluyver A J and Perquin C H C, Influence of oxygen on alcoholic fermentation, Biochem Z 266 (1933) Abbruzzese C, Duarte M Y, Paponetti B and Toro L, Biological and chemical processing of low-grade manganese ore, Min Eng 3 (1990) Srimekanond A, Thangevelu V J and Madgwick J C, Thermophilic bacterial leaching of manganese dioxide, J Ind Microbiol 10 (1992) Veligo F, Beolichini F, Gasbarro A, Toro L, Ubaldini S and Abbruzzese C, Batch and semi-continuous tests in bioleaching of manganiferous minerals by heterotrophic mixed microorganisms, Int J Miner Procecss 50 (1997)

8 TRANS. INDIAN INST. MET., VOL. 57, NO. 5, OCTOBER Silverman M P and Munoz F E, Fungal attack on rock: solubilization and altered infra-red spectra, Science 169 (1970) Ghiorse W C and Ehrlich H L, Microbial biomineralization of iron and manganese. In : (eds) Skinner, H C W, and Fitzpatrick R W, Biomineralization. Process of iron and manganese in modern and ancient environments. Catena Supplement 21, Catena, Verlag, Crelingen, Germany (1992) Madgwick J C, Bioleaching of manganese dioxide tailings, Biohydrometallurgical Technol, Vol.I, (eds) Torma, A E, Wey, J E, and Lakshmanan, V L (1993)