Abstract A strain of Aspergillus niger isolated from a metal-contaminated soil was able to grow in the presence of cadmium, chromium, cobalt, copper, and unusually high levels of nickel on solid (8.0 mM) and in liquid (6.5 mM) media. This fungus removed >98% of the nickel from liquid medium after 100 h of growth but did not remove the other metals, as determined by inductive- ly coupled plasma spectroscopy. Experiments with non- growing, live fungal biomass showed that nickel removal was not due to biosorption alone, as little nickel was bound to the biomass at the pH values tested. Further- more, when the protonophore carbonyl cyanide p-(triflu- oremetoxy) phenyl hydrazone (FCCP) was added to the actively growing fungus nickel removal was inhibited, supporting the hypothesis that energy metabolism is es- sential for metal removal. Analytical electron microsco- py of thin-sectioned fungal biomass revealed that metal removed from the broth was localized in the form of small rectangular crystals associated with the cell walls and also inside the cell. X-ray and electron diffraction analysis showed that these crystals were nickel oxalate dihydrate. Introduction Nickel (Ni) is widely used in industrial processes such as electronics manufacturing and metal plating and is found in wastewater from oil refineries and mines. From 1987 to 1993, nearly 27 million pounds of nickel were re- leased to water and land, primarily from nickel smelt- ing/refining and steelworks industries (US EPA 1995). Once released to the environment nickel readily forms complexes with many ligands, making it more mobile than most heavy metals. While nickel is an essential ele- ment at low concentrations for many organisms, it is tox- ic at higher concentrations. As little as 0.34 μM Ni inhib- its the growth of Escherichia coli (Abelson and Aldous 1950), whereas concentrations between 0.1 and 1.6 mM have been shown to inhibit the growth of various fungi (Adiga et al. 1961; Gadd 1993; Kumar et al. 1992; Mohan and Sastry 1984). Because of its toxicity, drink- ing water standards mandate nickel concentrations below 0.1 mg/l (1.7 μM). Ubiquitous soil and marine microorganisms play a major role in the fate of heavy metals in the environment (Gadd 1993). Fungi are known to tolerate and detoxify metals by several mechanisms including valence trans- formation, extra- and intracellular precipitation, and ac- tive uptake (Ashida 1965; Birch and Bachofen 1990; Joho et al. 1995; Tebo 1995; Volesky 1988). Recently, several fungi have been shown to precipitate such metals as cobalt, copper, zinc, manganese, and strontium as ox- alate, citrate, or pyruvate crystals (Gadd 1999; Gharieb et al. 1998; Sayer and Gadd 1997; Sayer et al. 1997). Passive metal removal or biosorption is also common among microorganisms (Al-Asheh and Duvnjak 1995; Kapoor and Viraraghaven 1998a; Mogollon et al. 1998; Tobin et al. 1984; Volesky 1988; Yetis et al. 1998). The high surface-to-volume ratio of microorganisms and their ability to detoxify metals are among the reasons mi- croorganisms are being considered as a potential alterna- tive to synthetic resins for remediation of dilute solutions of metals and solid wastes (Kapoor and Viraraghavan 1997, 1998a, b; Kapoor et al. 1999). We have isolated from a polluted natural environment a filamentous fungus of the genus Aspergillus that was not only able to tolerate unusually high levels of Ni on solid and in liquid media, but was also able to remove nickel from solution by precipitating it on the cell wall as nickel oxalate crystals. A. Magyarosy · R.D. Laidlaw · D.S. Clark · J.D. Keasling ( ✉ ) Department of Chemical Engineering, University of California, Berkeley, CA 94720–1462, USA e-mail: keasling@socrates.berkeley.edu Tel.: +510-642-4862, Fax: +510-643-1228 R. Kilaas · C. Echer Lawrence Berkeley National Laboratory, Berkeley, CA 94720–1462, USA Appl Microbiol Biotechnol (2002) 59:382–388 DOI 10.1007/s00253-002-1020-x ORIGINAL PAPER A. Magyarosy · R.D. Laidlaw · R. Kilaas · C. Echer D.S. Clark · J.D. Keasling Nickel accumulation and nickel oxalate precipitation by Aspergillus niger Received: 3 October 2001 / Revised: 18 March 2002 / Accepted: 2 April 2002 / Published online: 8 May 2002 © Springer-Verlag 2002