The role of acidophilic microorganisms in tetrahedrite dissolution and their contribution to acid mine drainage (AMD) formation remains poorly understood. Herein, the dissolution of tetrahedrite mediated by a moderately thermophilic consortium (MTC) enriched and acclimated from acidic mine sediments was investigated. MTC significantly accelerated the dissolution of tetrahedrite. After 60 days of bio-dissolution, the concentrations of Cu2+, Zn2+, AsT and SbT in leachate reached 2.26 g/L, 16.48 mg/L, 348.07 mg/L, and 22.88 mg/L respectively. MTC mobilized 52.15% copper from tetrahedrite, which was 6-fold higher than abiotic control (7.41%). The dissolution of tetrahedrite occurs through oxidative attack by Fe3+, which decomposes the mineral, while MTC reoxidizes the generated Fe2+ to Fe3+ and oxidizes sulfur species to sulfate, thereby enabling sustained oxidative dissolution. SEM-EDS revealed that MTC significantly enhanced mineral oxidation, causing microbial attachment and corrosion pits. However, XRD and XPS indicated that prolonged leaching induced a passivation layer dominated by jarosite, S0/S2- and Sb2O5. The MTC drives Fe3+ to oxidize As(Ⅲ)-O to As(V)-O. High-throughput sequencing analysis showed that Acidithiobacillus caldus, uncultured_archaeon_g__Aplasma, and Sulfobacillus thermotolerans were the dominant species through the bio-dissolution process. PICRUSt2 analysis showed enrichment of genes encoding copper efflux pumps (cusCBA), arsenic/antimony transport ATPases, and detoxification pathways, while arsenate reductase was downregulated to minimize toxic As3+ generation. This study provides novel insights into the bio-dissolution process of tetrahedrite mediated by acidophilic microorganisms, as well as highlights the potential of tetrahedrite in the generation of AMD.

The wastewater generated during sisal processing contains significant amount of organic matter, which will result in organic pollution of water bodies if discharged into environment directly. Temperature governs efficiency of organic degradation and power output of microbial fuel cells (MFCs) by regulating electroactive microbial activity and interfacial charge transfer. Considering the typical temperature ranges during the seasons of sisal processing, sisal wastewater was first treated with MFCs at 25 degrees C and 35 degrees C. Power density (PD) and chemical oxygen demand (COD) removal efficiency were 114.67 mW m- 2 and 92.15% respectively for MFC operated at 25 degrees C. However, a significant reduction of PD (72.92 mW m- 2) and COD removal rate (80.03%) were occurred when temperature elevated to 35 degrees C. The Nyquist plot results show that the resistance at 25 degrees C is lower than that at 35 degrees C, indicating that increase of temperature hinders electron transfer. At 25 degrees C, anode was dominated by Trichococcus (30.66%) and Geobacter (7.49%), while cathode harbored Trichococcus (17.19%) and Acetobacterium (7.28%). At 35 degrees C, the abundance of Trichococcus decreased to 13.81% in anode and 10.97% in cathode, with the anode further enriched in Geobacter (10.64%) and Petrimonas (8.68%). At 25 degrees C, the synergistic interaction between Trichococcus and Geobacter establishes an efficient electron transfer network, thereby enhancing the power density of MFC. Collectively, these results show that sisal wastewater can be effectively treated by MFC. This study explores the feasibility of using sisal wastewater as MFC substrate and elucidates the effects of temperature on MFC performance and organic removal efficiency.

Currently, whether robust anodic electroactive biofilms (EABs) are obtainable or not and how their electrochemical performances and metal remediation mechanisms during treating acidic mine drainage (AMD) remain largely unknown. Herein, a batch of acidic-cupric dual resistant anodic EABs were first enriched from sediment of AMD under different acidities, and Cu2+ removal mechanism during treatment of Cu2+-containing AMD in membrane-free microbial fuel cell (MFC) was explored. Results shows that increased acidity inhibited electrochemical performance of anodic EABs. The maximum output voltages of MFC with the EABs enriched at pH7.0, 5.5, 4.5, 3.5, and 3.0 were 505 ± 20 mV, 495 ± 10 mV, 353 ± 21 mV, 305 ± 4 mV, and 206 ± 13 mV respectively with an external resistance of 1000 Ω, which were only slightly inhibited in presence of 50 mg L-1 Cu2+ and recovered immediately after the absence of cupric stress. High power density (97.25 mW m-2) and high Cu2+ removal (92.47%) was achieved for the robust EABs enriched at pH3.0 in presence of 50 mg L-1 Cu2+. Mechanism analyses confirmed that Cu2+ was adsorbed by functional groups C-O-C and C-O on the surface of anodic EABs, and the removal of Cu2+ was co-precipitated as CuS and Cu2O. Significant differences on microbial community structure of these robust anodic EABs enriched under different acidities were observed. Alicyclobacillus and Thiomonas were significantly enriched in the robust anodic EABs enriched at pH3.0. This study provided novel insights to enrich robust EABs for developing bioelectrochemical technology for AMD remediation in future.

As bioleaching proceeds, the accumulation of hazardous substances (for example, organic matter) is detrimental to the survival of microorganisms. Microorganisms can cooperate with each other to resist the harsh environment. Unfortunately, little is known about how archaea improve bacterial growth and activity to cope with environmental stress. Here, bioleaching of sulphide ore (chalcopyrite and pyrite) was carried out by co-culture and pure culture of Leptospirillum ferriphilum and Ferroplasma acidiphilum under organic matter. The results showed that the copper and ferric extraction in the co-culture system were increased by 31.5 % and 31.7 % compared to the pure culture system of L. ferriphilum, respectively. In addition, L. ferriphilum grown better in the co-culture system compared with that in the pure culture system. Importantly, the results revealed that expression level of genes involved in oxidative phosphorylation and stress resistance were significantly upregulated in L. ferriphilum in the co-culture system compared to the pure culture system. This study is of great significance in gaining insights into the mechanisms of interactions between bioleaching microorganisms in extreme environments, and provides useful insights into how to improve bioleaching performance.

Anode material plays a pivotal role in increasing the performance of microbial fuel cells (MFCs). In this study, macroporous-mesoporous carbon derived from palm fiber (PF) as sustainable anode material was easily prepared via H3PO4 activation followed by carbonization. The MFC with palm fiber derived anode achieved a maximum power density of 718.15 mW m-2, which was 3.89 times higher than that of carbon cloth anode (146.78 mW m-2). P element was doped on PF anode by H3PO4 activation, which significantly improved the anodic hydrophilicity. The specific surface area (SSA), microporous structure, and P content of PF anode were significantly increased while O content was decreased when carbonization temperature increased from 500°C to 900°C. Collectively, PF anode was characteristic with high electrochemical performance and facile fabrication, which exhibiting great application potentials as an ideal MFC anode material in future.

Lactarius hatsudake is a common ectomycorrhizal edible mushroom in Pinus massoniana forests, and has important ecological and potential economic values. However, there are only a few reports on the establishment of Pinus massoniana–Lactarius hatsudake symbiosis. Here, we isolated a new strain of L. Lactarius hatsudake (GX01) from a local masson pine forest and established its ectomycorrhizal symbiosis with the P. massoniana. Potato dextrose agar (PDA) medium was optimal for the growth of L. hatsudake GX01. The saffron-to-brown ectomycorrhiza formed by L. hatsudake GX01 are usually bifurcated or coralloid shape, with a rod and a smooth surface, without emanating hyphae. The characteristic mantle and Hartig net structures of ectomycorrhizae were confirmed by microscope and scanning electron microscope (SEM). L. hatsudake GX01 can significantly promote the formation and development of lateral roots of P. massoniana seedlings during the early interaction. This study thus lays the foundation for subsequent study of the symbiotic molecular mechanism and application of P. massoniana–L. hatsudake symbiosis.

Heap bioleaching is one of the most promising technologies for extracting valuable metals from low-grade ores. However, the effects of permeability of the heap on microbial community and bioleaching efficiency remain unclear. In this study, heap bioleaching systems with different permeability were constructed. Despite the high content of larger particles had better permeability (0.25 cm/s) and oxygen transfer efficiency (0.14), there was a 25 % decrease in copper extraction compared with the moderate permeability group (81.2 %), while low permeability (0.025 cm/s) could cut the extraction in half (48.5 %). The fine profiles of microbial communities based on relatively and absolutely quantitative technologies suggested that permeability significantly affected microbial diversity, biomass, and composition. Microbial community evenness was crucial to improving extraction than biomass. Additionally, Thermoplasmatales except for Acidiplasma and Ferroplasma played vital roles in bioleaching. This study highlighted the delicate trade-off of particle size-mediated permeability for intensifying bioleaching efficiency of low-grade copper ores.

Methane-driven hexavalent chromium (Cr(VI)) reduction in a microbial fuel cell (MFC) has attracted much attention. However, whether the presence of sulfate (SO42-) affects the reduction of Cr(VI) is still lacking in systematic studies. This study involved constructing a MFC-granular sludge (MFC-GS) coupling system with dissolved methane (CH4) was used as the electron donor to investigate the effect of SO42- on Cr(VI) bioreduction, sludge characteristic, and functional metabolic mechanisms. When the SO42- concentration was 10 mg/L, the average removal rate of Cr(VI) in the anaerobic stage decreased to the lowest value (22.25 +/- 2.06%). Adding 10 mg/L SO42- obviously inhibited the electrochemical performance of the system. Increasing SO42- concentration weakened the fluorescence peaks of tryptophan and aromatic proteins in the extracellular polymeric substance of sludge. Under the influence of SO42-, Methanothrix_soehngenii decreased from 14.44% to 5.89%. The relative abundance of methane metabolic was down-regulated from 1.47% to 0.98%, while the sulfur metabolic was upregulated from 0.09% to 0.21% when SO42- was added. These findings provided some reference for the treatment of wastewater containing Cr(VI) and SO42- complex pollutants in the MFC-GS coupling system.

Water pollution is a global issue that has drastically increased in recent years due to rapid industrial development. Different technologies have been designed for the removal of pollutants from wastewater. However, most of these techniques are expensive, generate new waste, and focus solely on metal removal instead of metal recovery. In this study, novel facultative exoelectrogenic strains designated Castellaniella sp. A5, Castellaniella sp. B3, and Castellaniella sp. A3 were isolated from a microbial fuel cell (MFC). These isolates were utilized as pure and mixed culture inoculums in a bioelectrochemical system (BES) to produce bioelectricity and treat simulated industrial wastewater. A single-chamber MFC inoculated with the mixed culture attained the highest electricity generation (i.e., 320 mW/m2 power density and 3.19 A/m2 current density), chemical oxygen demand removal efficiency (91.15 ± 0.05%), and coulombic efficiency (54.81 ± 4.18%). In addition, the BES containing biofilms of the mixed culture achieved the highest Cu, Cr, and Cd removal efficiencies of 99.89 ± 0.07%, 99.59 ± 0.53%, and 99.91 ± 0.04%, respectively. The Cr6+ and Cu2+ in the simulated industrial wastewater were recovered via microbial electrochemical reduction as Cr3+ and Cu0, respectively. However, Cd2+ precipitated as Cd (OH)2 or CdCO3 on the surface of the cathodes. These results suggest that a mixed culture inoculum of Castellaniella sp. A5, Castellaniella sp. B3, and Castellaniella sp. A3 has great potential as a biocatalyst in BES for heavy metals recovery from industrial wastewater.

The impact of nickel (Ni2+) on the performance of anodic electroactive biofilms (EABs) in the bioelectrochemical system (BES) was investigated in this study. Although it has been reported that Ni2+ influences microorganisms in a number of ways, it is unknown how its presence in the anode of a BES affects extracellular electron transfer (EET) of EABs, microbial viability, and the bacterial community. Results revealed that the addition of Ni2+ decreased power output from 673.24 ± 12.40 mW/m2 at 0 mg/L to 179.26 ± 9.05 mW/m2 at 80 mg/L. The metal and chemical oxygen demand removal efficiencies of the microbial fuel cells (MFCs) declined as Ni2+ concentration increased, which could be attributed to decreased microbial viability as revealed by SEM and CLSM. FTIR analysis revealed the involvement of various microbial biofilm functional groups, including hydroxyl, amides, methyl, amine, and carboxyl, in the uptake of Ni2+. The presence of Ni2+ on the anodic biofilms was confirmed by SEM-EDS and XPS analyses. CV demonstrated that the electron transfer performance of the anodic biofilms was negatively correlated with the various Ni2+ concentrations. EIS showed that the internal resistance of the MFCs increased with increasing Ni2+ concentration, resulting in a decrease in power output. High-throughput sequencing results revealed a decrease in Geobacter and an increase in Desulfovibrio in response to Ni2+ concentrations of 10, 20, 40, and 80 mg/L. Furthermore, the various Ni2+ concentrations decreased the expression of EET-related genes. The Ni2+-fed MFCs had a higher abundance of the nikR gene than the control group, which was important for Ni2+ resistance. This work advances our understanding of Ni2+ inhibition on EABs, as well as the concurrent removal of organic matter and Ni2+ from wastewater.

During bauxite flotation, flotation indexes decrease with increasing water circulation time, resulting in the discharge of bauxite flotation wastewater as well as a waste of resources and great environmental safety risks. To determine how to return the wastewater back to the flotation process, this study aimed to identify the main components affecting the flotation indexes of flotation wastewater, and coagulation, biological and combined biological-coagulation methods were used to remove the components that affected the production index of wastewater. The results revealed that the accumulation of hydrolyzed polyacrylamide (HPAM) in circulating water reduced the flotation indexes. The combined biological-coagulation method was efficient and consisted of two steps: two-stage biological pretreatment to reduce the stability of HPAM in wastewater followed by coagulation treatment to remove HPAM. At an initial pH of 8, the biological coagulation treatment system stabilized after 4 days of continuous operation, and the treated water was subjected to a flotation test and attained good indexes: the proportion of alumina recovered increased from 62.80% to 68.52%, and the mass ratio of Al2O3 to SiO2 in the tailings decreased from 1.73 to 1.36. These indexes proved the feasibility of reusing the treated water for bauxite flotation. Microbial diversity analysis showed that Labrenzia played an important role in the biological pretreatment, and Gemmobacter, Stappia, Exiguobacterium, Pseudofulvimonas, and Acetoanaerobium played supporting roles. Based on the research results, a prototype reuse technology based on bauxite flotation wastewater treatment was established, and an industrial engineering application was developed through engineering design.

Smelting wastewater is characterized with high concentration of toxic heavy metals and high acidity, which must be properly treated before discharge. Here, bioelectrochemical system (BES) coupled with thermoelectric generator (TEG) was first demonstrated to simultaneously treat organic wastewater and smelting wastewater by utilizing the simulated waste heat that was abundant in smelting factories. By modulating the input voltage generated from simulated waste heat via TEG to 0, 1.0 and 2.0 V, almost all the Cu2+, Cd2+ and Co2+ in smelting wastewater were sequentially recovered with a respective rate of 121.17, 158.20 and 193.87 mg L-1 d(-1). Cu2+ was bioelectrochemically recovered as Cu-0. While, Cd2+ and Co2+ were recovered by electrodeposition as Cd(OH)(2), CdCO3 or Co(OH)(2) on cathodic surface. High throughput sequencing analysis showed that the microbial community of anodic biofilm was greatly shifted after successive treatment by batch-mode. Desulfovibrio (17.00%), Megasphaera (11.81%), Geobacter (10.36%) and Propionibacterium (8.64%) were predominant genera in anodic biofilm enriched from activated sludge in BES before treatment. After successive treatment by batchmode, Geobacter (34.76%), Microbacter (8.60%) and Desulfovibrio (5.33%) were shifted as the major genera. Economic analysis revealed that it was feasible to use TEG to substitute electrical grid energy to integrate with BES for wastewater treatment. In addition, literature review indicated that it was not uncommon for the coexistence of waste heat with typical pollutants (e.g. heavy metal ions and various biodegradation-resistant organic wastes) that could be treated by BES in different kinds of factories or geothermal sites. This study provides novel insights to expand the application potentials of BES by integrating with TEG to utilize widespread waste heat.

Acid mine drainage (AMD), characterized by a high concentration of heavy metals, poses a threat to the ecosystem and human health. Bioelectrochemical system (BES) is a promising technology for the simultaneous treatment of organic wastewater and recovery of metal ions from AMD. Different kinds of organic wastewater usually contain different predominant organic chemicals. However, the effect of different energy substrates on AMD treatment and microbial communities of BES remains largely unknown. Here, results showed that different energy substrates (such as glucose, acetate, ethanol, or lactate) affected the startup, maximum voltage output, power density, coulombic efficiency, and microbial communities of the microbial fuel cell (MFC). Compared with the maximum voltage output (55 mV) obtained by glucose-fed-MFC, much higher maximum voltage output (187 to 212 mV) was achieved by MFCs fed individually with other energy substrates. Acetate-fed-MFC showed the highest power density (195.07 mW/m2), followed by lactate (98.63 mW/m2), ethanol (52.02 mW/m2), and glucose (3.23 mW/m2). Microbial community analysis indicated that the microbial communities of anodic electroactive biofilms changed with different energy substrates. The unclassified_f_Enterobacteriaceae (87.48%) was predominant in glucose-fed-MFC, while Geobacter species only accounted for 0.63%. The genera of Methanobrevibacter (23.70%), Burkholderia-Paraburkholderia (23.47%), and Geobacter (11.90%) were the major genera enriched in the ethanol-fed-MFC. Geobacter was most predominant in MFC enriched by lactate (45.28%) or acetate (49.72%). Results showed that the abundance of exoelectrogens Geobacter species correlated to electricity-generation capacities of electroactive biofilms. Electroactive biofilms enriched with acetate, lactate, or ethanol effectively recovered all Cu2+ ion (349 mg/L) of simulated AMD in a cathodic chamber within 53 h by reduction as Cu0 on the cathode. However, only 34.65% of the total Cu2+ ion was removed in glucose-fed-MFC by precipitation with anions and cations rather than Cu0 on the cathode.

The effects of introducing M. sedula derivatives having different Cu2+-resistance on bioleaching capacity of a defined consortium (consisting of A. brierleyi DSM1651 and M. hakonensis HO1-1) were studied in column reactors at 70 °C. Introducing M. sedula copA mutant, a copper sensitive derivative, only had negligible effects on bioleaching. While introducing M. sedula ARS50-2, a Cu2+ resistant strain, substantially consolidated bioleaching process, with 27.77% more copper recovered after 58 d of bioleaching. Addition of M. sedula ARS50-2 likely enhanced the sulfur oxidation capacity of consortium after the 24th day under the Cu2+ stress. The majority of extreme thermoacidophiles were attached on minerals surface as indicated by quantitative PCR (qPCR) data. Successions of microbial community of extremely thermoacidophilic consortia that attached on surface of minerals were different from those in leachate. M. hakonensis HO1-1 was the dominant species attached on minerals surface in each column reactor throughout bioleaching process. The sessile M. sedula ARS50-2 remained as a major species till the 34th day. A. brierleyi DSM1651 was the most abundant planktonic species in leachate of each column reactor. These results highlight that higher Cu2+-resistance is a beneficial trait for extreme thermoacidophiles to process copper minerals.

Acid mine drainage (AMD) is a typical source of environmental pollution ascribing to its characteristics of high acidity and heavy metal content. Currently, most strategies for AMD treatment merely focus on metal removal rather than metal recovery. However, bioelectrochemical system (BES) is a promising technology to simultaneously remove and recover metal ions from AMD. In this study, both cupric ion and cadmium ion in simulated AMD were effectively recovered by BES inoculated with a novel exoelectrogen, Pseudomonas sp. E8, that was first isolated from the anodic electroactive biofilm of a microbial fuel cell (MFC) in this study. Pseudomonas sp. E8 is a facultative anaerobic bacterium with a rod shape, 0.43–0.47 μm wide, and 1.10–1.30 μm long. Pseudomonas sp. E8 can agglomerate on the anode surface to form a biofilm in the single-chamber MFC using diluted Luria-Bertani (LB) medium as an energy substrate. A single-chamber MFC containing the electroactive Pseudomonas sp. E8 biofilms has a maximum output voltage of 191 mV and a maximum power density of 70.40 mW/m2, which is much higher than those obtained by most other exoelectrogenic strains in the genus of Pseudomonas. Almost all the Cu2+ (99.95% ± 0.09%) and Cd2+ (99.86% ± 0.04%) in simulated AMD were selectively recovered by a microbial fuel cell (MFC) and a microbial electrolysis cell (MEC). After the treatment with BES, the high concentrations of Cu2+(184.78 mg/L), Cd2+(132.25 mg/L), and total iron (49.87 mg/L) in simulated AMD were decreased to 0.02, 0.19, and 0 mg/L, respectively. Scanning electron micrograph (SEM), energy dispersive X-ray spectrometry (EDXS) and X-ray diffraction (XRD) analysis indicate that the Cu2+ and Cd2+ in simulated AMD were selectively recovered by microbial electrochemical reduction as Cu0 (together with trace amounts of Cu2O) or Cd0 on the cathode surface. Collectively, data suggest that Pseudomonas sp. E8 has great potential for AMD treatment and metal recovery.

Extremely thermoacidophilic Crenarchaeota belonging to the order Sulfolobales, such as Metallosphaera sedula, are metabolically versatile and of great relevance in bioleaching. However, the impacts of extreme thermoacidophiles propagated with different energy substrates on subsequent bioleaching of refractory chalcopyrite remain unknown. Transcriptional responses underlying their different bioleaching potentials are still elusive. Here, it was first showed that M. sedula inocula propagated with typical energy substrates have different chalcopyrite bioleaching capabilities. Inoculum propagated heterotrophically with yeast extract was deficient in bioleaching; however, inoculum propagated mixotrophically with chalcopyrite, pyrite or sulfur recovered 79%, 78% and 62% copper, respectively, in 12 days. Compared with heterotrophically propagated inoculum, 937, 859 and 683 differentially expressed genes (DEGs) were identified in inoculum cultured with chalcopyrite, pyrite or sulfur, respectively, including upregulation of genes involved in bioleaching-associated metabolism, e.g., Fe2+ and sulfur oxidation, CO2 fixation. Inoculum propagated with pyrite or sulfur, respectively, shared 480 and 411 DEGs with chalcopyrite-cultured inoculum. Discrepancies on repertories of DEGs that involved in Fe2+ and sulfur oxidation in inocula greatly affected subsequent chalcopyrite bioleaching rates. Novel genes (e.g., Msed_1156, Msed_0549) probably involved in sulfur oxidation were first identified. This study highlights that mixotrophically propagated extreme thermoacidophiles especially with chalcopyrite should be inoculated into chalcopyrite heaps at industrial scale.

It is well acknowledged that the activities of activated sludge (AS) are influenced by seasonal temperature variation. However, the underlying mechanisms remain largely unknown. Here, the activities of activated sludge under three simulated temperature variation trends were compared in lab-scale. The TN, HN3-H, and COD removal activities of activated sludge were improved as temperature elevated from 20 °C to 35 °C. While, the TN, HN3-H, COD and total phosphorus removal activities of activated sludge were inhibited as temperature declined from 20 °C to 5 °C. Both the extracellular polymer substances (EPS) composition (e.g., total amount, PS, PN and DNA) and sludge index of activated sludge were altered by simulated seasonal temperature variation. The variation of microbial community structures and the functional potentials of activated sludge were further explored by metagenomics. Proteobacteria, Actinobacteria, Acidobacteria and Bacteroidetes were the dominant phyla for each activated sludge sample under different temperatures. However, the predominant genera of activated sludge were significantly modulated by simulated temperature variation. The functional genes encoding enzymes for nitrogen metabolism in microorganisms were analyzed. The enzyme genes related to ammonification had the highest abundance despite the changing temperature, especially for gene encoding glutamine synthetase. With the temperature raising from 20 °C to 35 °C. The abundance of amoCAB genes encoding ammonia monooxygenase (EC:1.14.99.39) increased by 305.8%. Meanwhile, all the enzyme genes associate with denitrification were reduced. As the temperature declined from 20 °C to 5 °C, the abundance of enzyme genes related to nitrogen metabolism were raised except for carbamate kinase (EC:2.7.2.2), glutamate dehydrogenase (EC:1.4.1.3), glutamine synthetase (EC:6.3.1.2). Metagenomic data indicate that succession of the dominant genera in microbial community structure is, to some extent, beneficial to maintain the functional stability of activated sludge under the temperature variation within a certain temperature range. This study provides novel insights into the effects of seasonal temperature variation on the activities of activated sludge.

Iron-oxidizing Acidithiobacillus spp. are applied worldwide in biomining industry to extract metals from sulfide minerals. They derive energy for survival through Fe2+ oxidation and generate Fe3+ for the dissolution of sulfide minerals. However, molecular mechanisms of their iron oxidation still remain elusive. A novel two-cytochrome-encoding gene cluster (named tce gene cluster) encoding a high-molecular-weight cytochrome c (AFE_1428) and a c4-type cytochrome c552 (AFE_1429) in A. ferrooxidans ATCC 23270 was first identified in this study. Bioinformatic analysis together with transcriptional study showed that AFE_1428 and AFE_1429 were the corresponding paralog of Cyc2 (AFE_3153) and Cyc1 (AFE_3152) which were encoded by the extensively studied rus operon and had been proven involving in ferrous iron oxidation. Both AFE_1428 and AFE_1429 contained signal peptide and the classic heme-binding motif(s) as their corresponding paralog. The modeled structure of AFE_1429 showed high resemblance to Cyc1. AFE_1428 and AFE_1429 were preferentially transcribed as their corresponding paralogs in the presence of ferrous iron as sole energy source as compared with sulfur. The tce gene cluster is highly conserved in the genomes of four phylogenetic-related A. ferrooxidans strains that were originally isolated from different sites separated with huge geographical distance, which further implies the importance of this gene cluster. Collectively, AFE_1428 and AFE_1429 involve in Fe2+ oxidation like their corresponding paralog by integrating with the metalloproteins encoded by rus operon. This study provides novel insights into the Fe2+ oxidation mechanism in Fe2+-oxidizing A. ferrooxidans ssp.

Thermophilic and lithoautotrophic archaea such as Metallosphaera sedula occupy acidic, metal-rich environments and are used in biomining processes. Biotechnological approaches could accelerate these processes and improve metal recovery by biomining organisms, but systems for genetic manipulation in these organisms are currently lacking. To gain a better understanding of the interplay between metal resistance, autotrophy, and lithotrophic metabolism, a genetic system was developed for M. sedula and used to evaluate parameters governing the efficiency of copper bioleaching. Additionally, adaptive laboratory evolution was used to select for naturally evolved M. sedula cell lines with desirable phenotypes for biomining, and these adapted cell lines were shown to have increased bioleaching capacity and efficiency. Genomic methods were used to analyze mutations that led to resistance in the experimentally evolved cell lines, while transcriptomics was used to examine changes in stress-inducible gene expression specific to the environmental conditions.