A moderately thermophilic mixed microbial culture for bioleaching of chalcopyrite concentrate at high pulp density

Abstract

Three kinds of samples (acid mine drainage, coal mine wastewater, and thermal spring) derived from different sites were collected in China. Thereafter, these samples were combined and then inoculated into a basal salts solution in which different substrates (ferrous sulfate, elemental sulfur, and chalcopyrite) served as energy sources. After that, the mixed cultures growing on different substrates were pooled equally, resulting in a final mixed culture. After being adapted to gradually increasing pulp densities of chalcopyrite concentrate by serial subculturing for more than 2 years, the final culture was able to efficiently leach the chalcopyrite at a pulp density of 20% (wt/vol). At that pulp density, the culture extracted 60.4% of copper from the chalcopyrite in 25 days. The bacterial and archaeal diversities during adaptation were analyzed by denaturing gradient gel electrophoresis and constructing clone libraries of the 16S rRNA gene. The results show that the culture consisted mainly of four species, including Leptospirillum ferriphilum, Acidithiobacillus caldus, Sulfobacillus acidophilus, and Ferroplasma thermophilum, before adapting to a pulp density of 4%. However, L. ferriphilum could not be detected when the pulp density was greater than 4%. Real-time quantitative PCR was employed to monitor the microbial dynamics during bioleaching at a pulp density of 20%. The results show that A. caldus was the predominant species in the initial stage, while S. acidophilus rather than A. caldus became the predominant species in the middle stage. F. thermophilum accounted for the greatest proportion in the final stage.

FIG 1

Copper extraction from chalcopyrite concentrate by a moderately thermophilic mixed culture adapted to leaching at different pulp densities.

FIG 2

Alignment of the 16S rRNA gene fragments of representative moderately thermophilic species such as L. ferriphilum, A. caldus, Sulfobacillus sp., and Ferroplasma sp. (The primer binding sites are highlighted by rectangles. Nucleic acid sequences are subdivided into blocks of 10 nucleotides by asterisks above them. Shading shows the similarity of nucleotides at the given site. The darker the shading is, the greater similarity is at that site. Uppercase letters under the nucleic acid sequence indicate that the given nucleotide is the same at the site for all organisms; lowercase letters indicate that fewer than 3 nucleic acid sequences were different from other sequences at the site.)

FIG 3

Verification analysis of universal primers for DGGE by PCR. (Lanes: 1, F. cupricumulans; 2, F. thermophilum; 3, F. acidiphilum; 4, L. ferriphilum; 5, A. caldus; 6, S. acidophilus; 7, S. thermosulfidooxidans.)

FIG 4

DGGE band patterns of the 16S rRNA genes of the moderately thermophilic culture during adaption at pulp densities of 2% (a), 4% (b), 6% (c), and 8% (d). (The arrows indicate the sequenced bands.) The letter d followed by a number at the top of the panels indicates experimental day.

FIG 5

Phylogenetic tree of the 16S rRNA gene sequences obtained by DGGE. Sequences detected by DGGE are highlighted by a triangle. The tree was constructed using sequences of comparable regions of the 16S rRNA gene sequences available in public databases. Neighbor-joining analysis using 1,000 bootstrap replicates was used to infer the tree topology.

FIG 6

Proportions of different species contained in a moderately thermophilic culture determined by DGGE during adaption at pulp densities of 2% (a), 4% (b), 6% (c), and 8% (d). (Black segments, S. acidophilus; light gray segments, A. caldus; dark gray segments, L. ferriphilum; white segments, F. thermophilum.)

FIG 7

Variations of copper concentration (a), pH (b), cell density (c), and concentrations of ferric iron and ferrous iron (d) in the solution during bioleaching of chalcopyrite concentrate with a moderately thermophilic culture at a pulp density of 20%.

FIG 8

Proportions of different species determined by real-time quantitative PCR on days 5 (a), 10 (b), 15 (c), and 20 (d) during bioleaching of chalcopyrite concentrate with a moderately thermophilic culture at a pulp density of 20%. (White segments, S. acidophilus; black segments, F. thermophilum; gray segments, A. caldus.)