Chalcopyrite (CuFeS2) hosts approximately 70 percent of the planet’s copper resources, which makes its efficient processing a central concern as high-grade reserves are rapidly depleted. The mineral’s problem is its extraordinarily stable Cu-Fe-S crystal lattice, which resists microbial attack and yields notoriously slow dissolution under mesophilic conditions, with copper recovery commonly stalling below 30 percent even after prolonged operation. The core obstacle is surface passivation. As bioleaching microbes oxidize the sulfide mineral, incomplete sulfur oxidation leaves behind coatings of elemental sulfur, polysulfides, and jarosite-type precipitates on the mineral surface and within the extracellular polymeric substance (EPS) layer of attached biofilms. These layers act as diffusion barriers, choking off mass transport and interfacial electron transfer between the microbes and the ore, and thereby suppressing dissolution kinetics. Overcoming this interfacial bottleneck has become the defining challenge of chalcopyrite bioleaching.

Researchers have previously tried to combat passivation with a range of physicochemical tricks: tuning solution pH and redox potential to discourage secondary precipitates, or adding catalysts such as silver ions, activated carbon, and organic electron mediators to promote electrochemical reactions at the mineral surface. While some of these approaches work in the laboratory, they are often constrained by cost, scalability, and unwanted side effects—silver ions, for instance, are toxic to the very bioleaching microorganisms they are meant to assist. Ecological regulation of the microbial community offers a complementary strategy, such as shifting the balance between sulfur-oxidizing and iron-oxidizing bacteria, but such approaches typically depend on exogenous inocula that decline in function over long-term operation. Phages, by contrast, are already natural residents of bioleaching environments. Earlier surveys of acid mine drainage and mine tailings have revealed abundant and diverse viral populations, and previous work by the same team showed that viruses help regulate microbial community assembly in copper mine bioleaching solutions.

In the new study, led by Zhaoyue Yang, Zhenghua Liu, and Huaqun Yin of Central South University, along with colleagues at Chengdu University, Wuhan University of Technology, the Central Metallurgical Research and Development Institute in Egypt, and Hunan Yama Biotechnology, the researchers set out to test whether deliberately introducing phages could tip the balance in favor of copper extraction. Phages were first induced from laboratory cultures of A. ferrooxidans using mitomycin C, which triggers dormant prophages embedded in the bacterial genome to enter their lytic cycle and burst their host cells. The released particles were concentrated with polyethylene glycol, purified by cesium chloride density gradient ultracentrifugation, and visualized by transmission electron microscopy, which revealed tailless, icosahedral capsids roughly 100 nanometers in diameter. Because conventional plaque assays are impractical in the strongly acidic media these acidophiles require, the phage dose was standardized relative to the host biomass used to produce the preparation.

The bioleaching experiments used chalcopyrite from the Dabaoshan mine in Guangdong, China, ground to 38–75 micrometers and suspended at 1 percent w/v pulp density in acidic medium at pH 2.0. Flasks were inoculated with an acid mine drainage-derived consortium dominated by Acidithiobacillus and incubated at 30 degrees Celsius. In the phage-treated group, the viral preparation was added on day 24, precisely the moment when visible precipitate accumulation signaled the onset of surface passivation and the layer was still loose enough to be accessible to phages. One day later, phage abundance in the treated flasks reached 9.42 × 10⁵ virus-like particles per milliliter—a striking 150-fold increase over the control group’s 6.26 × 10³. Virome sequencing of the inoculum identified two distinct viral operational taxonomic units, AfP_1732 and AfP_5388, carrying 12 and 11 predicted protein-coding genes respectively.

The impact on copper recovery was substantial. After 60 days, the phage-treated group achieved a copper leaching efficiency of 31.72 percent, compared with 20.43 percent in the untreated control—a relative improvement of 55.26 percent. Dissolved iron, sulfur, and copper concentrations all rose in the treated flasks, increasing by 30.27, 16.60, and 55.29 percent respectively. Most tellingly, phage addition rapidly reversed the kinetic slowdown that had set in by day 24: dissolved copper surged from 366.23 to 837.34 milligrams per liter in just 15 days, with an average copper release rate of 31.41 milligrams per liter per day, 2.65 times that of the control. Fitting the dissolution data to a shrinking core model showed that phages raised the product-layer diffusion rate constant 3.60-fold, which the authors estimate corresponds to a 72.2 percent reduction in the time needed to reach a given level of conversion.

Microscopic and spectroscopic analyses explained where this kinetic boost came from. Scanning electron microscopy showed that within three to nine days of phage addition, secondary precipitates and attached bacteria had visibly thinned on mineral surfaces, exposing fresh etch pits in the chalcopyrite. X-ray photoelectron spectroscopy of surface sulfur species revealed that elemental sulfur, which made up roughly 2.5 to 4.3 percent of surface sulfur in the control group at days 27 and 33, was entirely undetectable in the phage-treated flasks, and sulfate accumulations were similarly reduced. Fourier transform infrared spectroscopy, meanwhile, detected stronger amide and hydroxyl signals on treated surfaces, hinting at a shift in the extracellular polymeric layer toward more proteinaceous, more hydrophilic character. The authors attribute these changes partly to phage-encoded polysaccharide depolymerases and glycoside hydrolases, enzymes known to degrade the polysaccharide scaffolds that give biofilms their structural integrity, and partly to the capacity of phage capsids themselves to bind iron ions and serve as nucleation templates that draw mineral precipitates away from the ore surface.

The viral treatment also rewired the microbial ecology of the leaching system. Viromic profiling showed that 17 resident viral populations—distinct from the two phages in the inoculum—rapidly bloomed after phage introduction, and twelve of these were negatively correlated with Acidithiobacillus, indicating selective suppression of the dominant autotroph. In response, microbial diversity rose in both planktonic and mineral-associated communities within nine days. The relative abundance of mineral-associated Acidithiobacillus fell significantly while the heterotrophic genus Acidiphilium expanded, a shift the authors interpret as beneficial, since heterotrophs can consume organic metabolites that otherwise inhibit autotrophic leaching bacteria, echoing earlier findings that co-cultures of A. ferrooxidans and Acidiphilium acidophilum enhance iron oxidation and carbon fixation. Network analysis revealed altered co-occurrence patterns involving heterotrophic taxa such as Sphingomonas and Acinetobacter, and community assembly modeling indicated that phage pressure increased the role of homogeneous selection, pointing to more deterministic, phage-host-driven ecological dynamics. Metagenomic functional profiling reinforced the picture: genes of the Sox sulfur oxidation system, dissimilatory sulfur metabolism, and organic sulfur transformation were significantly enriched in the treated group, consistent with faster turnover of the elemental sulfur that builds passivation layers. A suite of antiphage defense systems, including Taranis, Shedu, and Ceres, was also enriched, marking an evolutionary arms race within the consortium. Notably, no sulfur metabolism genes were found in the introduced phages themselves, so the enhanced sulfur oxidation reflects community restructuring rather than viral auxiliary genes.

The authors argue that phage regulation carries two distinct advantages over conventional additives. First, host specificity means phages could in principle be deployed in stage-dependent fashion—promoting iron oxidizers early to regenerate ferric oxidant, and sulfur oxidizers later to strip elemental sulfur deposits—while simultaneously acting on both the microbial community and the mineral interface. Second, phage populations are self-amplifying and self-limiting: they multiply when hosts are abundant and fade when host density falls below the lytic threshold, potentially reducing the need for continuous supplementation that burdens chemical approaches such as silver ion catalysis, surfactants, activated carbon, L-cysteine, or ethylene thiourea, all of which carry cost, toxicity, or contamination concerns at industrial scale. The team is candid about the study’s limitations: the experiments were run at flask scale with a deliberately low 1 percent pulp density to enable clean observation of interfacial dynamics, and translating the approach to industrial heap or stirred-tank operations—characterized by high pulp densities, strong acidity, elevated ionic strength, and heterogeneous surfaces—will require further work on phage persistence, infectivity, and host encounter under those harsher conditions. Even so, the demonstration that a single phage amendment can loosen passivation layers, redirect sulfur metabolism, and meaningfully raise copper yields from the industry’s most stubborn ore opens an entirely new chapter in the ecological engineering of biomining, one in which the smallest inhabitants of acid mine drainage become tools for sustainable metal recovery.

Subject of Research: Phage-enhanced bioleaching of chalcopyrite ore

Article Title: Phages released from Acidithiobacillus ferrooxidans enhance chalcopyrite bioleaching by alleviating passivation and promoting sulfur turnover

Article References: Yang, Z., Liu, Z., Meng, D., Yang, Z., Hu, K., Yin, Z., Xia, L., Ibrahim, I. A., Xiao, X., Liu, X., & Yin, H. (2026). Phages released from Acidithiobacillus ferrooxidans enhance chalcopyrite bioleaching by alleviating passivation and promoting sulfur turnover. Advanced Biotechnology, 4(3), Article 29. https://doi.org/10.1007/s44307-026-00122-x

Image Credits: AI Generated

DOI: 10.1007/s44307-026-00122-x

Keywords: bioleaching, bacteriophages, chalcopyrite, copper extraction, Acidithiobacillus ferrooxidans, surface passivation, sulfur turnover, acid mine drainage, microbial communities, biomining, extracellular polymeric substances, mineral biotechnology