ARTICLE Non-Homogeneous Biofilm Modeling Applied to Bioleaching Processes Alvaro Olivera-Nappa, 1 Cristian Picioreanu, 2 Juan A. Asenjo 1 1 Centre for Biochemical Engineering and Biotechnology, Institute for Cell Dynamics and Biotechnology: A Centre for Systems Biology, University of Chile, Beauchef 850, Santiago, Chile; telephone: 56-2-9784716; fax: 56-2-6991084; e-mail: aolivera@ing.uchile.cl 2 Department of Biotechnology, Delft University of Technology, Delft, the Netherlands Received 17 December 2009; revision received 25 February 2010; accepted 3 March 2010 Published online 12 March 2010 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.22731 ABSTRACT: A two-dimensional non-homogeneous biofilm model is proposed for the first time to study chemical and biochemical reactions at the microorganism scale applied to biological metal leaching from mineral ores. The spatial and temporal relation between these reactions, microorganism growth and the morphological changes of the biofilm caused by solid inorganic precipitate formation were studied using this model. The model considers diffusion limitations due to accumulation of inorganic particles over the mineral sub- stratum, and allows the study of the effect of discrete phases on chemical and microbiological mineral solubilization. The particle-based modeling strategy allowed representation of contact reactions between the microorganisms and the insoluble precipitates, such as those required for sulfur attack and solubilization. Time-dependent simulations of chemical chalcopyrite leaching showed that chalcopyrite passivation occurs only when an impervious solid layer is formed on the mineral surface. This mineral layer hinders the diffusion of one kinetically determinant mineral-attack- ing chemical species through a nearly irreversible chemical mechanism. Simulations with iron and sulfur oxidizing microorganisms revealed that chemolithoautotrophic bio- films are able to delay passivation onset by formation of corrosion pits and increase of the solid layer porosity through sulfur dissolution. The model results also show that the observed flat morphology of bioleaching biofilms is favored preferentially at low iron concentrations due to preferential growth at the biofilm edge on the surface of sulfur-forming minerals. Flat biofilms can also be advanta- geous for chalcopyrite bioleaching because they tend to favor sulfur dissolution over iron oxidation. The adopted model- ing strategy is of great interest for the numerical representa- tion of heterogeneous biofilm systems including abiotic solid particles. Biotechnol. Bioeng. 2010;106: 660–676. ß 2010 Wiley Periodicals, Inc. KEYWORDS: bioleaching; biomining; chalcopyrite passiva- tion; mathematical model; flat biofilm morphology Introduction Copper mineral leaching at very low pH with the aid of biomining microorganisms is a widely known hydrome- tallurgical technique. The process involves oxidation of solid copper or copper iron sulfides (Cu 2 S, CuFeS 2 , etc.) to release into solution the desired copper ions (Cu 2þ ), together with other metallic ions, and forming in acidic conditions diverse soluble and insoluble sulfur compounds as a byproduct (S 0 , SO 2 4 ,S 2 O 2 3 , etc.) (Co ´ rdoba et al., 2008a; Rohwerder et al., 2003; Sand et al., 1995, 2001; Schippers and Sand, 1999; Suzuki, 2001; Watling, 2006). Typically, mineral oxidation is mainly caused by attack of ferric ions (Fe 3þ ) present in the solution, which are formed in turn by chemical oxidation of Fe 2þ by dissolved oxygen (O 2 ) or by biochemical oxidation of Fe 2þ by chemolithoautotrophic microorganisms. The most important mesophilic microbial species for copper biomining are Acidithiobacillus ferroox- idans, A. thiooxidans, and Leptospirillum ferrooxidans. A. ferrooxidans is the most versatile, given its ability to obtain energy by oxidizing Fe 2þ or elemental sulfur (S 0 ) at very low pH values (Stott et al., 2003). The protons generated by sulfur oxidation maintain the acidic environ- ment for the microorganisms to thrive. A. thiooxidans can only oxidize elemental sulfur, while L. ferrooxidans can only oxidize Fe 2þ (Stott et al., 2003). Ferrous ion and sulfur are oxidized by microorganisms mainly using oxygen as the final electron acceptor. Some microorganisms are able to use other electron acceptors in the absence of oxygen, and they can even oxidize sulfur using ferric ions (Brock and Gustafson, 1976; Donati et al., 1997; Hansford and Vargas, 2001; Lundgren and Silver, 1980; Ohmura et al., 2002; Pronk et al., 1992; Rawlings, 2005; Sugio et al., 1985), but the energy yield of these biotransformations is too small to be taken into account in aerobic environments. Fe 2þ is soluble and thus readily available to the cells, but sulfur is insoluble, and its oxidation involves a surface reaction that requires cell attachment to Correspondence to: A. Olivera-Nappa Contract grant sponsor: Millennium Scientific Initiative of the Government of Chile Additional Supporting Information may be found in the online version of this article. 660 Biotechnology and Bioengineering, Vol. 106, No. 4, July 1, 2010 ß 2010 Wiley Periodicals, Inc.