Heavy metal tolerance of fungi

Minerals Engineering, Vol. 14, No. 5, pp. 499-505, 2001

Pergamon

© 2001 Elsevier Science Ltd All rights reserved 0892-6875/01/$ - see front matter

0892-6875(01)00037-1

HEAVY METAL TOLERANCE

OF FUNGI*

M . V A L I X , J . Y . T A N G a n d R. M A L I K Department of Chemical Engineering, The University of Sydney, NSW 2006, Australia E-mail: mvalix @chem.eng.usyd.edu.an (Received 11 December 2000; accepted 17 February 2001)

ABSTRACT The tolerance of fungi strains including Penicillium funiculosum, Aspergillus foetidus, Penicillium simplicissimum for different heavy metals, which could be leached, from nickel laterite ores (Ni, Co, Fe, Mg and Mn) was studied. These strains were exposed to heavy metals up to 2000 ppm. The tolerant strains were selected by repeated subcutturing in petri dishes with increasing metal concentration in the medium. The degree of tolerance was measured from the growth rate in the presence of the various heavy metals and compared to a control, which contained no heavy metals. It appears that Penicillium funiculosum and Aspergillus foetidus were the most tolerant to the heavy metals and exhibited strong growth often exceeding the control. Penicillium simplicissimum showed the least tolerance particularly for Ni and Co. A growth pattern, which was consistent for each strain under various heavy metals, was observed as a function of time. The growth pattern of the fungi exhibited a lag, retarded, similar and enhanced rate of growth in the presence of heavy metal relative to the control. The similarity in the pattern appears to suggest the tolerance development or adaptation of the fungi for heavy metals. © 2001 Elsevier Science Ltd. All rights reserved.

Keywords Oxides ores; bioleaching INTRODUCTION Nickel laterite ores, have been shown to be amenable to leaching by heterotrophic microorganism and their metabolic products (Valix et. al, 2000, Boseker, 1985, Tzeferis et.al., 1994). Development and optimisation of such a process will depend on the isolation of microorganism, which would be tolerant to various conditions, including temperature, pH and heavy metal concentrations during the leaching process. Understanding the interaction between heavy metals and microorganisms has perhaps attracted the most interest to researchers due to its application in metallurgy and decontamination of the environment. Heavy metals in small concentrations can induce morphological changes and then destroy the microorganism cell. At relatively high concentrations heavy metals act as a general protoplasmic poison, inducing denaturation of proteins and nucleic acid (Avakyan, 1994). Despite the natural actions of heavy metal on the microorganism, they demonstrate an ability to survive by adapting and mutating under various conditions including high concentrations of heavy metals. The control of such operations will allow strain to be tailor made for specific applications and would greatly improve the biological application of these microorganisms to mineral processing and for environmental protection. Isolation of strains with tolerance to Ni has been conducted by Boseker, 1985, Tzeferis et.al., 1994, Tzeferis, 1994, with favourable results. However in leaching nickel laterites ores, various metals, including * Presented at Minerals Engineering 2000, Cape Town, South Africa, November 2000

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Co, Fe, Mg, Mn, Cu, A1, Cr, Zn are also solubilised including sulfur species. Tzeferis (1994), observed fungi strains adapted to tolerate Ni concentration up to 4000 ppm could not survive in 10% of laterite pulp density, which only contains about 750 ppm of Ni. This appears to be associated with the cumulative toxic actions of other dissolved heavy metals and suggest the relative value of isolating strains with tolerance for all the heavy metals involved in the leaching. The objective of this study is to identify and isolate fungi strains with particular tolerance toward heavy metals, which could be leached from nickel laterite ores. This was conducted with the specific aims of establishing the relative toxicity of the leached metals to the fungi strains, the effect of metals and their concentration on the growth of fungi strain and identifying the strains which demonstrates greater resistance to heavy metal toxicity.

EXPERIMENTAL Microorganism and growth conditions Fungal strains including Penicillium funiculosum, Aspergillus foetidus and Penicillium simplicissimum were tested for their resistance and growth in the presence of various concentrations of metals: Ni, Co, Fe, Mg and Mn. These microorganisms were chosen based on their ability to produce acids such as citric acid and oxalic acid, which were previously found to be effective in chelating metals from laterite nickel ore. Penicillium funiculosum, Aspergillus foetidus and Penicillium simplicissimum were grown in a Czapek Yeast Extract Agar (CYA) media which consisted of 1L distilled water, 1.0g/1 K2HPO4, 3.0g/1 NaNO3, 0.5g/l MgSO4.7H20, 0.5g/l KCI, 0.01g/l FeSO4.7H2 O, 30.0g/1 sucrose, 5.0g/1 yeast extract, 15.0g/l agar, and trace elements. The strains were then allowed to grow at temperatures between 25 -32 °C in an incubator. Preparation of media and heavy metals The strain selection involved exposing the various fungus strains with synthetic leachate metal solution produced from dissolved NiC1.6H20, COC12.6H20, Fe(NO3)2.6H20, MgSO4.7H20, and MnCI2. The media were previously sterilised in an autoclave at 121°C for 15 minutes. These were allowed to cool down to around 60°C and poured into the petri plates for the cultures growth. The sterilisation was conducted to ensure cross contamination with other organism do not occur. Strain isolation Strain isolation was conducted through a series of repeated subculturing of the fungi exposed to different concentrations of metals including Ni, Co, Fe, Mg and Mn in plate test. Isolation and acclimatisation were conducted in petri dish, 8 cm in diameter, containing the growth medium with metal concentration from 500 to 2000 ppm. The plates were inoculated with a 4-mm circular extract of growing fungi strain. The plates were incubated at 25-32 °C between 9 to 18 days to establish their growth rate at the lower metal concentration (500-ppm). The growth was monitored by measuring the spread of the culture from the point of inoculation or centre of the colony. Strains demonstrating growth are sampled and exposed to another petri dish with a higher metal concentration. Tolerance was measured from the growth of the fungi in the presence of metal divided by the growth in the same period in the absence of metal.

RESULTS AND DISCUSSION Metal concentrations of leached laterite ores Nickel laterite ores, which are of interest in this study, include limonite, nontronite, and weathered and fresh saprolites. Limonite is distinguished by high iron content and saprolites by the high silicate content. Nontronite is the phase between limonite and nontronite. The data reported in Table 1 gives an estimate of the potential metal concentrations of leachate solution from laterite ores, if all metals are to dissolve. However, it is anticipated that metal concentrations would be lower than these, as complete extraction does not occur, as commonly found in practice. Two-pulp densities 50 and 100 g/L are provided for comparison.

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TABLE 1 The maximum leachable metals of various laterite ores with pulp density. Ore A: fresh saprolite, B: weathered saprolite, C: limonite, D: nontronite Elements

Ni Co Fe M8 Mn Cu AI Cr Zn

A Ppm

Pulp Density: 508/I, Ore Types B C ppm ppm

D Ppm

A Ppm

1345 30 3250 9750 45 I0 2100 240 10

1990 60 6550 7500 60 3 1380 355 15

1285 70 6550 1450 110 3 1645 3790 1.5

2690 60 6500 19500 90 20 4200 480 20

765 100 23650 600 515 5 2200 930 15

Pulp Density: lOOg~ Ore Types C B ppm ppm 3980 120 13100 15000 120 6 2760 710 30

1530 200 47300 1200 1030 10 4400 1860 30

D Ppm 2570 140 13100 2900 220 6 3290 7580 3

Growth phase of fungi The relative toxicity of various metals on the growth rate of Penicillium simplicissimum, AspergiUus foetidus, Penicillium funiculosum is shown in Figures 1 to 3. The concentration of the metals to which the fungi were exposed to was kept constant at 500 ppm, for this particular test. To allow measurements of the effect of heavy metals on the growth rate of the fungi a tolerance index was defined. This index was taken as the measured growth in the presence of metal divided by the growth of the fungi in the same period in the absence of metal. The common definition of relating the tolerance index to the heavy metal concentrations which is found to reduce the growth rate by 50% of the control, was not used in this study. Although this may form some basis for comparison, it may be misleading, as the tolerance development of the fungi, as found in this study, was also dependent on time of training. It is evident that the presence of heavy metals retards the growth of most of the fungi strains, shown from tolerance index of less than 1, in Figures 1 to 3. The metal toxicity, have been reported to have a tendency to increase with molecular weight (Avakyan, 1994). It would thus be expected that the order of toxicity would be Mg, Mn, Fe, Ni, Co. This was confirmed from previous studies which showed that Ni was one of the most toxic for Aspergillus niger (Avakyan, 1974) and strains of PeniciUium (Kaltwasser et. al, 1980).

1.0

0.8

•~ ¢ 0.6

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500 ppm NI, PeniclUlums. 500 ppm Co,PenlcNUums. 500 pore Fe,Penic#liums. 500 ppm Mg,PenlciUiums. 500 ppm Mn,Penicilllum s.

0.2-

0.0 ",r = 0

I

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2

4

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6 8 T~ (a~)

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12

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Fig. 1 Tolerance index of Penicillium simplicissimum in 500 ppm of Ni, Co, Fe, Mg and Mn at 25 °C.

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1.6 1.4 1.2 i

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500 ppm Co,A.foeUdus 500 ppm Fe,A.foetidus 500 ppm Mg,A.foeUdus 500 ppm Mn,A.foetidus

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2

4

6

8

10

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Time (days) Fig.2 Tolerance of Aspergillusfoetidus in 500 ppm of Ni, Co, Fe, Mg and Mn at 25 °C.

1.6-

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Fig.3 Tolerance of Penicilliumfuniculosum in 500 ppm of Ni, Co, Fe, Mg and Mn at 25 °C. This study, however, appear to suggest a different order in the relative toxicity of heavy metals. The relative toxicity of the heavy metals reflected by the tolerance index was found to vary with time of exposure and type of strain. In general it appears Penicillium simplicissimum and Penicillium funiculosum, shows higher tolerance towards Ni and Co and lower tolerance for Fe, Mn and Mg. Whereas Aspergillus foetidus appears to have better tolerance for Mg, Mn and Ni, less for Fe and Co. The favourable tolerance for Ni observed for the Penicillium strains in this study is comparable to that reported by Boseker, 1985. The growth pattern, for each of the fungi under the various metals, as shown in Figures 1 to 3, show predictable patterns. This pattern is similar for each fungi strain and appears to reflect the tolerance development of the fungi, which could be used as a basis for training these microorganisms. The growth pattern of the fungi in the presence of metal, as depicted in Figure 4, can be characterised by five stages: a)

Heavymetaltoleranceof fungi

503

lag phases which occurs at the beginning of the inoculation, where very little growth occurs, b) rapid growth rate but absolute growth is suppressed relative to control (tolerance level less than 1) c) decline in growth rate, d) levelling of the tolerance index indicative that the rate of growth of fungi with metals and control are similar and finally e) rapid growth with absolute growth exceeding the control (tolerance index greater than 1):

b

c

\i

Tolerance Index

I I I I I I I I I I I I I

d

e

/ I.~end; a lag phase b rapid growth c retarded growth d similar growth e enhanced

Time Fig.4

The growth phases of fungi in the presence of heavy metals relative to control grown in the absence of heavy metals.

Tolerance training of fungi The effect of increasing the metal concentrations on the tolerance development of the fungi are summarised in Table 2. The fungi PeniciUium simplicissimum and Aspergillus foetidus was shown to thrive in the presence of heavy metals. Although it is anticipated that some heavy metals are necessary for the growth of fungi, the required levels are often low quantities (< 200 ppm). The concentrations used in this study is considered to be toxic to these fungi. After developing tolerance at 500 ppm, Penicillium simplicissimum and Aspergillus foetidus were shown to grow more vigorously, particularly at the higher concentration of heavy metal up to 2000 ppm. This is reflected by the higher tolerance index in phase d, and the reduction in the lag phase in phase a. This is in contrast to Penicilliumfuniculosum, which was shown to have lower tolerance with increasing concentration of metals. Previously reported tolerance of similar PeniciUium and Aspergillus strains (Hashem et. al., 1994, Kansoh et. al., 1998, and Okamoto et. al., 1977) are typically in the region of 200 to 1000 ppm of Ni and Co. Some of these strains were acclimatised within only a short period of time, typically 3 to 4 days. The growth phase of the fungi as observed in this study (see Figures 1 to 2), indicate that early harvesting may result in a strain which has not fully adapted to the heavy metals. This is characterised in phase c, which shows a reduced rate of growth relative to the control. These may suggest the reason for poorer tolerance developed by similar strains. Although further tests are required to confirm this finding, it poses an intriguing hypothesis for strain development. Strains demonstrating higher tolerance have also been reported (Boseker, 1994, El Sharouny et. al, 1988 and Tzeferis, 1994). Some of these strains have been developed over a longer acclimatisation period (12-14 days), which supports the merit of longer adaptation period. It does indicate that heavy metals are toxic to microorganisms, however subculturing and the adaptation period appear to have an influence on degree of tolerance developed by the fungi. The relative toxicity of the heavy metals for each strain becomes obvious at the higher concentrations. A general order, which fits all strain, is difficult to determine from the results. Aspergillus foetidus and PeniciUiumfuniculosum showed similar relative tolerance index to the order of toxicity at the high and low

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levels of heavy metals. The adaptive nature of the Aspergillus strain, particularly for Ni, in this study was contradictive to poorer tolerance previously reported for this type of microorganism (Boseker, 1985). Penicillium simplicissimum, although its tolerance index was maintained at the same value on exposure to metal concentration of Ni from 500 to 2000 ppm, its relative tolerance with respect to the other metals appear lower. It would suggest that Aspergillus foetidus would have a better potential in being used in the biological leaching of laterite based on it tolerance of the desired metals, Ni and Co, being selectively extracted. It should be stated however that further test are required to assess the fungi strains to fully establish their applicability in leaching laterite ores. These include the amount of metal taken up by the fungi, temperature tolerance and the efficiency in producing the required organic acids. TABLE 2 Effect of heavy metal concentration on growth characteristics of fungi in phase a and d Metal Concentration (ppm)

Nickel 500 1500 2000 Cobalt 500 1500 2000 Iron 500 1500 2000 Magnesium 500 1500 2000 Manganese. 500 1500 2000

Growth Phase Characteristics

Aspergillusfoetidus

Penicillium simplicissimum

Penicillium funiculosum

Phase d Tolerance index

Phase a Lag (days)

Phase d Tolerance index

Phase a Lag (days)

Phase d Tolerance index

Phase a Lag (days)

0.7 0.84 0.86

1 1 1

0.8 0.8 1.25

1 1 3

0.8 0.35 0.22

1 2 2

0.62 1.0 1.2

1 1 I

0.8 1.3

1 0

0.8 0 0

3 No growth No growth

0.6 0.95 1.0

1 1 1

0.6 0.8 1.3

1 1 1

0.75 0.7 0.75

3 2 1

0.58 1.25 1.15

1 1 1

0.6 1.25 1.25

1 0 1

1.3 0.9 0.85

3 2 1

0.6 1.15 1.22

1 1 1

0.75 1.25 1.2

1.0 0.84 0.75

1 2 1

CONCLUSIONS Fungi growth was found to be suppressed by heavy metal, but subculturing of strain resulted in mutation and adaptation of fungi within 8 days where enhanced growth in the presence of heavy metals was observed. Among the strains acclimatised Penicillium simplicissimum and Aspergillus foetidus generated the most tolerant species, showing enhanced growth even at high concentrations (2000 ppm) of heavy metals. Penicillium funiculosum was the most sensitive and the least tolerant, in particular for Ni and Co metals. Relative toxicity of the heavy metals measured from the tolerance index was found to vary with time and the type of strains. No general order was found which could fit all the strains. Fungi growth phase, which was observed from the tolerance index of the fungi with time upon exposure to heavy metals, was found to be a predictable pattern for each strain and metal concentrations. The growth phase was characterised by a lag, retarded, similar and enhanced rates compared to control which appear to reflect the tolerance development or adaptation of fungi in the presence of heavy metals. Overall, it appears tolerance of Penicillium simplicissimum and Aspergillus foetidus strains are promising and have the potential to be developed for leaching laterite ores.

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REFERENCES

Avakyan, Z.A, The Toxicity of Heavy Metals to Microorganism. Qatar University Science Journal, 1994, 14, 3-65. Avakyan, Z.A., Microbiology, Volume 2, Biology Series, 1974, 3-28. Bosecker, K., Leaching of Lateritic Nickel Ores with Heterotrophic Microorganisms in Fundamental and Applied Biohydrometallurgy: Proceedings of the Sixth International Symposium on Biohydrometallurgy, Vancouver, B.C., Canada, August 21-24, 1985, pp. 367-382. El-sharouny, H., Bagy, M. and EI-Shanwany A., Toxicity of Heavy Metals to Egyptian Soil Fungi. International Biodeterioration, 1988, 24, 49-64. Hashem A.R., and Bahkali A.H., Toxicity of Cobalt and Nickel to Fusarium Solani Isolated from Saudi Arabian Soil. Qatar University Science Journal, 1994, 14(1) 63-65. Kaltwasser H., and Frings, W., Nickel in the Environment, (ed J.O. Nriagu) John Wiley and Sons Inc., 1980, 463-491. Kansoh A., L. and EI-Shafei H., Multiple Heavy Metal Tolerance in Some Fungal and Bacterial Strains. The African Journal of Mycology and Biotechnology, 1998, 6(3),31--40. Okamoto, K, Suzuki, M., Fukami, M., Toda, S., and Fuwa, K., Uptake of Heavy Metals by a Copper Tolerant Fungus, Penicillium ochro-chloron. Agricultural Biological Chemistry, 1977, 41(1), 17-22. Tzeferis, P.G., and Agatzinin-Leonardou, S., Leaching of Nickel and Iron form Greek Non-Sulphide Nickeliferous Ores by Organic Acids. HydrometaUurgy, 1994, 36, 345-360. Tzeferis, P.G., Leaching of a Low Grade Hematitic Laterite Ore Using Fungi and Biologically Produced Acid Metabolites, International Journal of Mineral Processing, 1994, 42, 267-283. Valix, M., Usai F. and Malik, R., Fungal Bio-Leaching Of Low Grade Laterite Ores. Minerals Engineering, 2001, 14 (2).

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