Acidithiobacillus thiooxidans secretome containing a newly described lipoprotein Licanantase enhances chalcopyrite bioleaching rate

Abstract

The nature of the mineral-bacteria interphase where electron and mass transfer processes occur is a key element of the bioleaching processes of sulfide minerals. This interphase is composed of proteins, metabolites, and other compounds embedded in extracellular polymeric substances mainly consisting of sugars and lipids (Gehrke et al., Appl Environ Microbiol 64(7):2743-2747, 1998). On this respect, despite Acidithiobacilli-a ubiquitous bacterial genera in bioleaching processes (Rawlings, Microb Cell Fact 4(1):13, 2005)-has long been recognized as secreting bacteria (Jones and Starkey, J Bacteriol 82:788-789, 1961; Schaeffer and Umbreit, J Bacteriol 85:492-493, 1963), few studies have been carried out in order to clarify the nature and the role of the secreted protein component: the secretome. This work characterizes for the first time the sulfur (meta)secretome of Acidithiobacillus thiooxidans strain DSM 17318 in pure and mixed cultures with Acidithiobacillus ferrooxidans DSM 16786, identifying the major component of these secreted fractions as a single lipoprotein named here as Licanantase. Bioleaching assays with the addition of Licanantase-enriched concentrated secretome fractions show that this newly found lipoprotein as an active protein additive exerts an increasing effect on chalcopyrite bioleaching rate.

Fig. 1

Bidimensional SDS-PAGE of the sulfur batch culture secretome of A. thiooxidans. Identified proteins are highlighted with arrows and numerated according to: 1 Licanantase lipoprotein; 3 hypothetical protein Lferr_1276; 4 glyceraldehyde-3-phosphate dehydrogenase; 5 protein TolA; 6 pentapeptide repeat-containing protein, IcmE; 7 hypothetical protein Lferr_1859; 8 3-isopropylmalate dehydratase, large subunit; 9, 10 phosphate-selective porin O and P

Fig. 2

Bidimensional SDS-PAGE of the sulfur batch culture metasecretome of A. thiooxidans/A. ferrooxidans mixed culture. Identified proteins are highlighted with arrows and numerated according to: 2, 11, and 12 Licanantase lipoprotein; 13 phosphate-selective porin O and P; 14 hypothetical protein PSEEN2944; 15 cytochrome c biogenesis protein transmembrane region

Fig. 3

Amino acid sequence alignment of the putative lipoprotein from A. ferrooxidans ATCC 53993 (YP_002220838.1|) and the lipoprotein Licanantase from A. thiooxidans DSM 17318. Amino acid sequence identity of 96%

Fig. 4

Chalcopyrite bioleaching assays. Black triangle, 5× total sulfur secretome of A. thiooxidans; empty circle, 5× total sulfur metasecretome of A. thiooxidans/A. ferrooxidans; empty square, control with no addition of secretome; black diamond, control without inoculum. Left, percent Cu recovery; right, bacterial growth determinations

Fig. 5

Chalcopyrite bioleaching assays. Filled triangle, 5× >30 kDa subfraction of the total sulfur secretome of A. thiooxidans; empty circle, 5× <30 kDa subfraction of the total sulfur secretome of A. thiooxidans; empty diamond, control with the addition of A. thiooxidans heat-inactivated total secretome; empty square, control with no addition of secretome subfractions; filled diamond, control without inoculum. Left, percent Cu recovery; right, bacterial growth determinations