A strain of Leptospirillum sp. CC previously isolated from Akhtala polymetallic ore (Armenia) was studied. The main morphological and physiological characteristics of CC were revealed. The optimal growth temperature was 40 °C and optimal pH 1.5. A phylogenetic analysis based on 16S rRNA gene sequences (GenBank ID OM272948) showed that isolate CC was clustered with L. ferriphilum and possessed 99.8% sequence similarity with the strain L. ferriphilum OL12-2 (KF356024). The molar fraction of DNA (G + C) of the isolate was 58.5%. Bioleaching experiment indicates that L. ferriphilum CC can oxidize Fe(II) efficiently, and after 17 days, 44.1% of copper and 91.4% of iron are extracted from chalcopyrite and pyrite, respectively. The efficiency of L. ferriphilum CC in pyrite oxidation increases 1.7 times when co-cultivated with At. ferrooxidans ZnC. However, the highest activity in pyrite oxidation shows the association of L.ferriphilum CC with heterotrophic Acidocella sp. RBA bacteria. It was shown that bioleaching of copper and iron from chalcopyrite by association of L. ferriphilum CC, At. ferrooxidans ZnC, and At. albertensis SO-2 in comparison with pure culture L. ferriphilum CC for 21 days increased about 1.2 and 1.4–1.6 times, respectively.

Bioleaching of Cu from the copper concentrate of Armanis gold-bearing polymetallic ore (Armenia) was investigated. The main objective was revealing high active bacteria and their association, as well as optimizing the bioleaching process with their application to ensure the most efficient recovery of copper from the tested concentrate. To obtain optimal bacterial associations, bottom-up and top-down approaches were used. Bioleaching of copper concentrate was carried out using pure cultures of iron- and sulfur-oxidizing bacteria and their mixed culture, as well as indigenous bacterial consortium. Comparative studies of copper bioleaching by mixed cultures of Acidithiobacillus caldus, Leptospirillum ferriphilum CC, Sulfobacillus thermosulfidooxidans 6, and indigenous consortium Arm of iron-oxidizing bacteria were performed. At the beginning of bioleaching, the amounts of extracted copper by mixed culture and Arm consortium were equal; afterward, between 20–27 days, the Arm indigenous consortium showed significantly higher activity in terms of copper extraction. In parallel, mineralogical and liberation analyses of feed material and bioleaching residues were performed.

A new strain of Leptospirillum sp. Ksh-L was isolated from a dump-bioleaching system of the Kashen copper-molybdenum mine (South Caucasus). Ksh-L is an obligate chemolithoautotroph, capable of oxidizing ferrous iron (Fe²⁺). Cells are Gram-negative and vibrio- or spirillum-shaped of a 0.5–3 µm size. The optimal conditions for the growth are 35 °C and pH 1.6–1.8. Cu²⁺ and Zn²⁺ have different effects on the oxidizing ability of the Leptospirillum sp. Ksh-L culture depending on the phase of growth and concentration of Fe²⁺. Under the conditions of gradually increasing the concentration of copper in the medium, during 4–5 successive subculturing experiments, it was possible to obtain an adapted culture of Leptospirillum sp. Ksh-L, capable of growing in the medium in the presence of up to 400 mM Cu²⁺. A bioleaching experiment indicates that Ksh-L can efficiently oxidize chalcopyrite. However, the bioleaching of copper from chalcopyrite by Leptospirillum ferrodiazotropum Ksh-L increased about 1.8 times in association with At. thiooxidans ATCC 19377. Phylogenetic analysis based on 16S rRNA gene sequences (GenBank ID ON226845) shows that strain Ksh-L forms a single cluster into Group III. The strain possesses 99.59%, 99.52%, and 96.60% sequence similarity with the strains YTW-96-06, YTW-66-06, and Leptospirillum ferrodiazotrophum 5C in Group III, respectively.

Chloride ions can enhance the bioleaching of copper minerals, yet most biomining microorganisms are highly sensitive to chloride and cannot survive or colonize mineral surfaces in saline environments. Chloride tolerance varies among acidophilic iron-oxidizing bacteria, but the concentrations at which they remain active are generally too low to permit the industrial use of seawater. Therefore, identifying highly chloride-tolerant leaching microorganisms and studying their bioleaching potential in chloride-containing systems is of utmost importance. This study investigated chloride tolerance and adaptability of bacteria from different genera, with a focus on Sulfobacillus thermosulfidooxidans subsp. asporogenes 41, a moderately thermophilic strain that can oxidize both Fe (II) and reduced inorganic sulfur compounds (RISCs). This dual activity makes it advantageous for bioleaching by facilitating sulfur removal, generating acidity, and preventing mineral passivation. Comparative experiments on the bioleaching of pyrite and chalcopyrite demonstrated that adaptation to 0.3 M NaCl enhanced the chloride tolerance of S. thermosulfidooxidans subsp. asporogenes 41. The adapted strain exhibited significantly improved copper extraction under saline conditions compared with the native culture. Maximum copper recovery was achieved at 0.4 M NaCl, highlighting the potential of chloride-adapted moderate thermophiles for biomining applications in saline environments. In contrast the minimal inhibitory concentration for Acidithiobacillud ferrooxidans Dr was 0.005 M (causing 41.2% inhibition), while Leptospirillum ferriphilum CC was unaffected by lower concentrations (0.01–0.02 M) and only showed severe inhibition (86.5%) at 0.1 M NaCl, defining its minimal inhibitory concentration (MIC) at 0.05 M.

The microbial ecology of acid mine drainage (AMD) systems in Armenia, with a long mining history, remains unexplored. This study aimed to characterize the microbial diversity and functional potential of AMD in the Syunik region and to isolate novel microorganisms with biotechnological value. A comprehensive analysis of the microbial communities’ structure of Kavart abandoned, Kapan exploring mines effluent, and Artsvanik tailing was conducted. Metagenomics revealed bacterial-dominated communities, comprising Pseudomonadota (previously “Proteobacteria”) (68–72%), with site-specific variations in genus abundance. A high abundance and diversity of metal resistance genes (MRGs), particularly for copper and arsenic, were identified. Carbohydrate-active enzyme (CAZy) analysis showed a dominance of GT2 and GT4 genes, suggesting a high potential for extracellular polymeric substances (EPS) production and biofilm formation. A novel strain of iron-oxidizing bacteria Arm-12 was isolated that shares only ~90% similarity with known Leptospirillum type species, indicating it may represent a new genus without culturable representatives. The strain exhibits enhanced copper extraction from concentrate. This study provides the first metagenomic insights into Armenian AMD systems and tailing, revealing a unique community rich in metal resistance and biofilm-forming genes. The isolation of a novel highly effective iron-oxidizer Arm-12 highlights the potential of AMD environments as a source of novel taxa with significant applications in biomining and bioremediation processes.

The oxidation of sulfide minerals under atmospheric conditions often results in the generation of acid mine drainage (AMD), which is characterized by low pH and elevated concentrations of dissolved metals, including heavy metals. In this study, a molecular biology-based approach was applied for the first time to analyze bacterial communities in AMD and tailing samples from the Syunik Region of Armenia. The microbial community analysis revealed that Proteobacteria was the dominant phylum across all three samples, followed by Actinobacteria and Bacteroidetes. The diversity at the genus level was found to correlate with the chemical composition of the samples. Metagenomic profiling identified a variety of metal resistance genes (MRGs), with the most abundant being related to resistance against copper (copA, copC, copF, copG, copR, ruvB), iron (acn), arsenic (arsC, arsT, pstB), silver (silA), and mercury (merA, merR1). A novel iron-oxidizing bacterium, designated Arm-12 (GenBank: PP389931), was isolated from AMD. It showed only 90% similarity to known Leptospirillum species, suggesting it may belong to a new genus. This strain is thermophilic, with optimal growth observed at 45°C and pH 1.8–2.0. Toxicity tests showed that its iron-oxidizing activity was inhibited in the presence of various metals, with the following order of toxicity: Mo > Cr > Co > Ni > Zn > Cu. Kinetic studies demonstrated Arm-12’s high potential for biogenic regeneration of ferric iron (Fe³⁺), underscoring its applicability in bioleaching processes.