Biomineralization of Copper: Solutions for Waste Remediation and Biomining

C.R. Ashby, S.A. Thompson,and T.C. Crusberg

Department of Biology & Biotechnology, Worcester Polytechnic Institute, Worcester MA 01609, Phone: (508)831-5472, Fax: (508)831-5936


The fungus Penicillium ochro-chloron is able to extract copper from aqueous solutions and form insoluble copper precipitates within the matrix of fungal mycelia. The formation of these complexes is probably a detoxification mechanism used by the organism to deal with the potentially lethal concentrations of heavy metals. Metal immobilization occurs external to the cells but within the mycelia when the solubility products of copper phosphate and copper oxalate are exceeded. This process may be exploited in biomining to remove and recover copper and perhaps other heavy metals that have become solubilized in pit mine lakes.

Keywords: metal immobilization, aqueous solutions, heavy metals


Large amounts of heavy metals are released into the environment each year from industrial wastewater through inefficiencies built into the technological activities used directly in the processing of metals. The metallic species released into the environment tend to be maintained there indefinitely, circulating and eventually accumulating throughout the food chain, posing a serious threat to the environment and public health.

There are numerous methods currently employed to try to remove and recover the metals polluting our environment. These include such techniques as chemical oxidation and reduction, membrane separation, liquid extraction, carbon adsorption, ion exchange, electrolytic treatment, electroprecipitation, coagulation, flotation, evaporation, hydroxide and sulfide precipitation, vitrification, and crystallization (McFeters, 1990). These methods differ in their effectiveness and cost. They also often tend to treat the metal totally as waste without the possibility of recycling.

One potential alternative to existing technologies for removing heavy metals from industrial wastewater is biosorption by microbial cells. It is well known that metal ions are involved in all aspects of microbial life. Small concentrations of metal ions are needed within microorganisms to stabilize a range of biological structures, act as enzyme co-factors,and serve many other functions. When essential metals are at high concentrations however, they become toxic to microorganisms (Hughes and Poole, 1989). In order to deal with the toxicity some microorganisms have developed mechanisms including one by which they convert the metal ions to insoluble metal complexes. These metal complexes form as precipitates external to the cytoplasm of the microorganism, rendering the metal harmless. This normal biological process may be a potentially effective procedure for solving and controlling major industrial environmental problems associated with metals (Kandori, et al., 1993; Volesky, 1990), including biomining of dilute (about 200 mg/L Cu) solutions found in pit mine lakes. It was our objective to study one potential solution to this problem.

In literature concerning the biosorption of heavy metals, a variety of different organisms have shown the ability to accumulate an array of different metals. Lead and cadmium have been effectively removed from solutions by species of brown algae such as Ascophyllum and Sargassum. Mycelia of the fungi Rhizopus and Absidia have been shown to be excellent biosorbents for lead, cadmium, copper, zinc, and uranium. Saccharomyces species have the ability to uptake silver, cadmium, cobalt, and copper. Gold, cadmium, chromium, copper, iron, and manganese can be taken up by Bacillus species (Volesky & Holan, 1995). There are many more types of fungi, algae, yeast, and bacteria that have the ability to sequester metal ions.

For our experimentation, we chose to study the fungus P. ochro-chloron (obtained from the American Type Culture Collection) and its uptake of copper. Copper is an essential element for plants and animals and is found in great abundance in nature (Alloway, 1995). It occurs in the crust of the earth at an average concentration of approximately 50 mg/kg, principally as a sulfide (Morre, 1991). Copper is not as toxic to living organisms as some other metals, but its extensive use and increasing levels in the environment are cause for concern. The situation found in the Berkeley Pit Mine in Butte, Montana, is one serious example of copper pollution. Mining operations conducted there between 1876 and 1979 contaminated over 25 billion gallons of water that had accumulated in the Berkeley Pit since the mine was shut down. This water contains dissolved copper at 172 mg/L at a pH of 2.85 or lower, vastly different from the allowable concentration of that ion whose discharge permitted according to federal regulations which specify a maximum of 0.15 mg/L copper at a pH between 6 and 9 (Atlas and Bartha, 1987).

The organism chosen for this study, the fungus Penicillium ochro-chloron, is one of the best examples of microbial metal tolerance (Edwards, 1990). P. ochro-chloron is able to tolerate high concentrations of heavy metals, specifically copper (Volesky, 1990). The fungus can grow in saturated CuSO4 and can tolerate up to 5,000 mg/L of copper (Stokes and Lindsay, 1979). P. ochro-chloron can be easily grown, maintained, and harvested in the laboratory which makes it a very good choice to study.

The mechanism associated with the biosorption of single species of heavy metal ions using microorganisms as biosorbents is affected by several factors which include the specific surface properties of the organism and the physiochemical parameters of the solution such as temperature, pH, initial metal ion concentration, and biomass concentration (Sag and Kutsal, 1995). The mechanism at work with certain Penicillium species is probably a passive binding of metal ions. Passive, rather than active, uptake of metal ions is suspected because of both the high speed of uptake, which would preclude metabolic activity, and the observation of no loss of uptake ability with the death of the cell (Galun, et al., 1983).

There are believed to be a variety of ways in which cells take up metal ions and these include ion exchange, chelation, adsorption by physical forces, and ion entrapment in inter- and intrafibrillar capillaries and spaces of the structural polysaccharide network as a result of the concentration gradient and diffusion through cell walls and membranes (Volesky and Holan, 1995). Fungal pigments have also been implicated in metal uptake and resistance by some fungal species (Volesky, 1990). There are several chemical groups that could attract and sequester metals in biomass (Sag and Kutsal, 1995). The cell wall of fungi consists of amino or nonamino-polysaccharides. These polysaccharides play an important part in the uptake of metal from solution. The amino and carboxyl groups and the nitrogen and oxygen of the peptide bond could be available for characteristic coordination bonding with metal ions (Sag and Kutsal, 1995). Furthermore, the hydroxyls in polysaccharides are believed to attract metal ions. The amino, amido, sulfhydryl, and carboxyl groups in proteins are also able to attract and thus chelate metal ions (Volesky & Holan, 1995). However this does not seem like a probable uptake mechanism in P. ochro-chloron since fungal cell walls are often poor in proteins (Sag and Kutsal, 1995). It is important to remember that the presence of some functional groups does not guarantee their accessibility for sorption. Other factors such as steric or conformational hindrances or other barriers may be present in the cell.

Also, pH greatly affects the uptake of metal ions by fungi (Poole, 1995). Biosorption of heavy metals usually leads to the acidification of solutions (Volesky and Holan, 1995). The different pH binding profiles for heavy metal ions could be due to the nature of the chemical interactions of each metal with microbial cells and are related to the isoelectric point of the cell (Sag and Kutsal, 1995). At pH values above the isoelectric point, there is a net negative charge on the cells and the ionic state of ligands such as carboxyl, phosphate, and amino groups will be such as to promote reaction with the metal cations. As the pH is lowered, however, the overall surface charge on the cells becomes positive, which will inhibit the approach of positively charged metal cations (Sag and Kutsal, 1995). When the surface becomes uncharged, the resulting pH represents an isoelectric point with the lowest solubility of metal ions. While this situation is very favorable for the deposition of metals, it makes the study of binding sites more difficult (Volesky and Holan, 1995).

Studies have shown that the sorption of some metals tends to cause the formation of insoluble micro-precipitates. The formation of these precipitates is a detoxification mechanism used by the organism in order to deal with the potentially toxic concentrations of metal (Hughes and Poole, 1989). The mechanism by which these precipitates form is more complicated due to the fact that the collection of the metal species is not due to the straight sequestration mechanism (Volesky and Holan, 1995).

Many techniques are currently employed to recover metal ions embedded on or within organisms. Simple pH adjustment can lead to the stripping of bound metals for those metal ions that show a strong pH dependence in biosorption. For example, when the pH is dropped to 2.0, copper cations biosorbed by C. vulgaris at pH 5 are quantitatively released. If there is little pH dependence, specific ligands can be added to the solution and will form extremely stable complexes with the metal ions. This will effectively remove the metal from the organism (Stokes and Lindsay, 1979). Some researchers have also tried using physical methods, such as centrifugation and ultrasonication, to extract metals (Grainger and Lynch, 1984).

Materials and Methods

All solutions were made up with deionized water with aeseptic technique where appropriate. All chemicals were analytical reagent-grade quality. Penicillium ochro-chloron was employed as the model biotrap in these experiments because of its known ability of removing copper and other heavy metals from aqueous solutions. Using cornmeal agar as a medium (Difco), the P. ochro-chloron spores grew quickly and sporulated evenly on the plate. The fungal spores were two weeks old before they were suspended in sterile deionized water and transferred, using a Pasteur pipette, into a plastic conical centrifuge tube and stored in the refrigerator for up to two weeks before use.

Eight to ten drops of spore suspension was transferred into each of ten 300 ml plastic flasks containing glucose mineral salts (GMS) [18 g/L glucose, 2 g/L NaNO3, 0.5 g/L MgSO4 and KCl, 1 g/L KH2PO4, 0.1 g/L CaCl2, 0.01 g/L FeSO4 and 5 mL Tween 80]. After four days of growth at 300C and shaking at 300 RPM in a gyratory incubator, the 3-4 mm dia. beads were washed aeseptically with new minimal medium (NMM) [0.5 g/L KCl and MgSO4, 0.2 g/L NaNO3, 0.1 g/L CaCl2, and 1 g/L KH2PO4] supplemented with 1 g/L glucose and 200 mg/L Cu (as CuSO4) and incubated at 30oC at 250 RPM in a gyratory shaker for another four days.

The beads were then prepared for scanning electron microscopy and energy dispersive x-ray (EDX) chemical analysis. Having been washed in deionized water for at least one hour, the beads needed to be quick-frozen using petroleum ether at -700C. After removing the beads from the solution using tweezers, they were dropped into a test tube of petroleum ether kept at -700C in an ice block previously frozen in a Revco freezer. The beads froze instantly and either clung to the sides of the tube or fell to the bottom. The ether was carefully poured off into another tube held in a block of ice also at -700C. The beads were then quickly but carefully removed from the tube and placed into a sterile dish. Most beads cracked in half after immersion in the petroleum ether, but those that did not crack open needed to be cut in half with dissection scissors quickly to avoid thawing. The frozen beads were lyophilized in a Virtis freeze drier overnight. Using a graphite adhesive, the dried beads were mounted onto graphite coated aluminum stubs. Next, the samples were coated with carbon in a Varian vacuum evaporator to reduce the charging effect caused by the electrons of the SEM. Other samples were coated in a Fullam sputter coater with 200 A of Au/Pd 60:40. The samples were examined using a JEOL JSM 5600 scanning electron microscope and elemental analysis was done using a Noran energy dispersive x-rays (EDX) analysis unit fitted to the microscope.

Copper oxalate and copper phosphate were prepared by adding saturated solutions of either potassium oxalate or potassium phosphate respectively to one of 20,000 mg/L copper sulfate. The precipitates (one mL) were washed seven times with 13 mL deionized water for each wash by centrifugation. The wet precipitates were spread over the surface of a carbon-coated electron microscope stub and after drying ovrenight in a desiccator were coated with carbon as above.

Results and Discussion

A normal P. ochro-chloron bead grown in a non-metal containing solution is pale in color. Upon closer inspection of the bead using an SEM, a complex mesh of hyphae are observed criss-crossing through the interior of the bead. Separating the hyphae from the environment is a thin (5-10 mm) outer crust.

Fungal beads challenged with 200 mg/L copper, on the other hand, exhibited a bluish-green color. Using a JEOL JSM 5600 scanning electron microscope, the mycelia are evident with a central cavity (Fig. 1A, B). Small spheres embedded within the fungal mycelial beads are seen amongst the hyphae (Fig. 1B). In general, any small sphere found within 5-10 mm of the surface was classified as in the exterior of the bead. Beyond this point, the beads were deemed to be on the interior of the bead [refer to Fig. 1B]. The small spheres are either porous (Fig. 1C) or smooth (Fig. 1D). Through both SEM and EDX analyses, two different types of spheres were observed called mineralspheres with distinct textures. One mineralsphere showed the presence of both copper and phosphorous (Fig. 1C and Fig. 2A) and appeared to have a spongy, rough exterior. The other mineralsphere (Fig. 1D and Fig. 2B) also showed the presence of copper but lacked the characteristic phosphorous signal and exhibited a smooth exterior. These two mineralspheres were determined to be copper phosphate and copper oxalate, respectively. Fig. 3 shows the EDX analysis for the baseline EDX analysis for copper phosphate (upper spectrum) and copper oxalate (lower spectrum). In the copper oxalate spectrum, there is a small aluminum peak. As seen also in some of the fungal beads, this peak is due to a thinner graphite coating beneath the fungal bead, providing a greater exposure to aluminum.

Discussion and Conclusions

Copper shows a pronounced tendency to form complexes with inorganic and organic ligands. Around neutral pH, most of the inorganic copper in solution is present as complexes with carbonate, nitrate, sulfate, and chloride (Morre, 1991). Neutral ligands such as ammonia, ethylenediamine, and pyrimidine also form strong four-coordinated complexes. Copper has also been shown to form complexes such as copper oxalate (Dirsken, et al., 1990) and copper phosphate (Kandori, et al., 1993).

Dirsken et al. reported that copper oxalate particles grown by atomistic growth mechanisms usually resulted in smooth, hard, crystalline objects with a well-defined crystal habit. They also suggested that the particles were formed by an aggregation process because of the morphology of the copper oxalate produced. Through size and time analyses, they noticed that as time progressed further, the median size of the particles increased in a logarithmic fashion. Copper oxalate formation is believed to occur in P. ochro-chloron because the fungus produces oxalic acid through action of the enzyme oxaloacetase, which could render copper harmless to the organism as the insoluble compound copper oxalate (Moat and Foster, 1995; Olafson, 1994; Voet and Voet, 1995). Oxalate is then exported and reacts extracellularly with copper to produce copper oxalate.

Kandori et al. examined nickel phosphate particles to try to determine their formation and inner structure. They studied the crystallized form of Ni3(PO4)2 and noticed that the nickel phosphate particles exhibited a high porosity. The nickel phosphate mineralspheres that they obtained have the same porous-appearing exteriornoticed in the copper mineralspheres found within P. ochro-chloron.

Copper oxalate and copper phosphate have solubility products of Ksp = 4.43 x 10-10 and Ksp = 1.40 x 10-37, respectively (Fine and Beall, 1990; Lide, 1996). The theoretical concentration of Cu+2 in solution in the presence of solid copper oxalate is 0.013mg/L and copper phosphate is for all intents and purposes, insoluble.

P. ochro-chloron has the ability to remove and recover copper from solution. After further study, it may prove to be an effective means for controlling metals in the environment. However, more work is needed to determine how multiple species of metals can be dealt with by the fungus Penicillium ochro-chloron. It is at this time not possible to predict how this organism would respond to the multiplicity of effects which can occur when many different metals are present (Sag and Kutsal, 1995).


This research was supported by U.S.E.P.A. grant R82-3341-01-1.


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Figure 1. Scanning electron micrographs of fungal mycelia. A. 4 mm dia. bead showing hollow interior. B. A freeze-cracked 4 mm fungal bead showing mineralspheres within the interior. C. porous mineralsphere. D. Smoother mineralsphere.

Figure 2. A. EDX analysis of the mineralsphere in Fig. 1C. B. EDX analysis of mineralshphere in Fig. 1D.

Figure 3. EDX analysis of copper phosphate (upper spectrum) and copper oxalate (lower spectrum) prepared in this laboratory.