Isolation, Sphalerite Bioleaching, and Whole Genome Sequencing of Acidithiobacillus ferriphilus QBS3 from Zinc-Rich Sulfide Mine Drainage

Abstract

The genus Acidithiobacillus has been widely used in bioleaching, and novel strains in this genus, such as A. ferriphilus, have also been confirmed to possess bioleaching capabilities. In this study, an Acidithiobacillus ferriphilus strain, QBS3, was isolated from zinc-rich sulfide mine drainage using the gradient dilution method. QBS3 is a Gram-negative, 1.3 µm rod-shaped bacterium with small red colonies. It showed a high iron oxidation efficiency of 0.361 g/(L·h) and a sulfur oxidation efficiency of 0.206 g/(L·d). QBS3 has sphalerite bioleaching ability; using QBS3 for pure sphalerite bioleaching, 18.8% of zinc was extracted in 14 days at 1% pulp density. Whole genome sequencing was performed on QBS3. Functional prediction showed that 9.13% of the genes were involved in replication, recombination, and repair. Bioleaching-related genes were analyzed, including iron and sulfur oxidation genes, and carbon and nitrogen fixation genes. For iron oxidation, the Cyc2→RusA pathway and Iro→RusB pathway were found in QBS3. In terms of sulfur oxidation, QBS3 has an incomplete SOX system and lacks the SDO gene, but Rho and Trx may complement the SOX system, enabling QBS3 to oxidize sulfur. QBS3 has multiple sets of carbon fixation genes, and nitrogen fixation genes were also identified. A hypothetical sphalerite bioleaching model is proposed; this study provides a theoretical basis for the zinc sulfide ore bioleaching industry.

Keywords: Acidithiobacillus ferriphilus; bioleaching; ferrous oxidation; isolation; sphalerite; sulfur oxidation.

Figure 1

(A) Sampling site; (B) colony image; (C) bacterial cell SEM image; and (D) 16S rRNA gene phylogeny tree of QBS3 and seven closely related strains.

Figure 1

(A) Sampling site; (B) colony image; (C) bacterial cell SEM image; and (D) 16S rRNA gene phylogeny tree of QBS3 and seven closely related strains.

Figure 2

(A) Sulfur oxidation and growth curves of QBS3 in 10 g/L sulfur; (B) iron oxidation curve of QBS3 in 9 g/L Fe(II); (C) QBS3 cultured in 9K medium with the addition of 10 g/L sulfur; and (D) QBS3 cultured in 9K medium with the addition of 9 g/L Fe(II).

Figure 2

(A) Sulfur oxidation and growth curves of QBS3 in 10 g/L sulfur; (B) iron oxidation curve of QBS3 in 9 g/L Fe(II); (C) QBS3 cultured in 9K medium with the addition of 10 g/L sulfur; and (D) QBS3 cultured in 9K medium with the addition of 9 g/L Fe(II).

Figure 3

(A) Zinc leaching curve of 1% to 4% pulp sphalerite with QBS3; (B) sulfite content change curve; (C) total iron content change curve; (D) pH change curve; (E) SEM image of sphalerite before bioleaching; and (F) after bioleaching.

Figure 3

(A) Zinc leaching curve of 1% to 4% pulp sphalerite with QBS3; (B) sulfite content change curve; (C) total iron content change curve; (D) pH change curve; (E) SEM image of sphalerite before bioleaching; and (F) after bioleaching.

Figure 4

(A) SEM image and EDS analysis results of QBS3 cells attached to sphalerite; (B) biofilm on sphalerite.

Figure 5

Circular genome map of A. ferriphilus QBS3. From outside to center: genes on the direct strand, genes on the complementary strand, tRNAs (blue), rRNAs (purple), GC content (yellow means higher than average, blue means lower), GC-skew. (A) Chromosome; (B) plasmid; (C) Gene annotation.

Figure 6

The genomic structures of genomic islands (A) and prophages (B) in the genome of A. ferriphilus QBS3.

Figure 7

(A) eggNOG function classification; (B) GO function classification of the QBS3 genome.

Figure 8

(A) DDH calculations between QBS3 and typical strains from the genus Acidithiobacillus; (B) ANI heatmap between QBS3 and other A. ferriphilus strains.

Figure 9

(A) Core gene rarefaction curve, Pan Gene (blue), Core gene(red); (B) whole genome phylogenetic tree of A. ferriphilus; and (C) synteny analysis result of strains QBS3, GT2, and YL25.

Figure 10

The putative mechanism model of sphalerite bioleaching by A. ferriphilus QBS3. The dissolution of sphalerite results from the oxidative attack of ferric ions (Fe³⁺) on the surface. In this interfacial redox reaction, ferric ions are reduced into ferrous ions (Fe²⁺) by the electrons from sphalerite, while the elements of Zn and S of sphalerite are gradually released into the bioleaching solution from the surface. The released soluble zinc ions (Zn²⁺) are extracted and thus achieve the industrial purpose of zinc metallurgy. The released sulfur may generate various reduced inorganic sulfur compounds (RISCs), such as hydrogen sulfide (H₂S), polysulfur (S_n), sulfite (SO₃²⁻), thiosulfate (S₂O₃²⁻), etc., due to the complex reaction of sulfur chemistry. The role of A. ferriphilus in sphalerite bioleaching is to oxidize these ferrous ions and various RISCs. The oxidation of Fe²⁺ leads to the regeneration or recycling of the oxidant of Fe³⁺. The oxidation of RISCs results in the transformation of insoluble or inhibiting intermediates into the final highly soluble product of sulfate (SO₄²⁻), thereby promoting the progress of sphalerite bioleaching. Meanwhile, the oxidation of spharelite by A. ferriphilus provides energy for the fixation of carbon dioxide (CO₂) and nitrogen (N₂) for the growth and reproduction of this chemoautotroph. The iron oxidation system of A. ferriphilus consists of downhill and uphill electron transfer pathways. The downhill pathway is from the oxidation of extracellular Fe²⁺ by Cyc2 or periplasmic Fe²⁺ by Iro, and then successively transfers electrons to rusticyanins (RusA and RusB), Cyc1, the aa₃ complex, and O₂. The uphill pathway pushed by the transmembrane gradient proton is bifurcated from rusticyanins (RusA and RusB), and then successively transfers electrons to CycA1, reverse bc₁ complex, quinone pool, reverse NDH, and NADPH. The sulfur oxidation system of A. ferriphilus mainly contains SQR, Rho, SoxYZ, Trx, SoxB, TQO, TerH, etc. The various extracellular RISCs enter the periplasmic space via the outer membrane or pore channels. SQR catalyzes H₂S to produce sulfane sulfur (S_n) and puts an electron into the quinone pool. SoxYZ binds the sulfur with the help of Rho and is then oxidized by Trx to produce SoxYZ-thiosulfate adduct and bring an electron to the aa₃ complex via CycA2. SoxB splits this adduct to generate the final product of sulfate. Sulfur and sulfite can non-enzymatically and reversibly be placed into thiosulfate. TQR catalyzes thiosulfate to produce tetrathionate (O₃²⁻SSSSO₃²⁻) and puts electrons into the quinone pool. TetH hydrolyzes tetrathionate to produce thiosulfate, sulfur, and sulfate. The forward bc₁ complex extracts electrons from the quinone pool and then transfers them to the aa₃ complex via Hip to reduce oxygen into water. The bo₃ complex directly extracts electrons from the quinone pool to reduce oxygen. Under the actions of various respiratory components, the transmembrane proton gradient is formed and thus drives ATP synthetase to produce ATP. The carbon fixation is realized by the Calvin cycle with the energies of ATP and NADPH. The nitrogen fixation is performed by the nitrogenase system (NifHDK and FDR) under the energy of ATP and NADPH.